Methods of reducing incidence of mortality in fowl

ABSTRACT

The disclosure relates to methods for decreasing incidence of mortality in fowl through administering isolated microorganisms to the fowl. In particular aspects, the disclosure provides methods of decreasing incidence of mortality in fowl through administration of a Clostridium sp. and a Clostridium cluster XIV bacterium to the fowl. The disclosure further relates to isolated microorganisms and compositions comprising the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. Utility application under 35 U.S.C. § 111that claims priority pursuant to 35 U.S.C. § 120: (I) as a ContinuationApplication, to U.S. patent application Ser. No. 16/042,369, filed Jul.23, 2018, which is a Continuation-In-Part Application of InternationalApplication No. PCT/US2017/028015, filed on Apr. 17, 2017, which itselfclaims the benefit of priority to U.S. Provisional Application No.62/323,305, filed on Apr. 15, 2016; U.S. Provisional Application No.62/335,559, filed on May 12, 2016; and U.S. Provisional Application No.62/425,480, filed on Nov. 22, 2016; (II) this application also claimsthe benefit of U.S. Provisional Application No. 62/574,031, filed onOct. 18, 2017; the entirety of each and every one of the aforementionedapplications is herein expressly incorporated by reference in theirentirety.

FIELD

The present disclosure relates to isolated and biologically puremicroorganisms that have applications, inter alia, in the farming offowl. The disclosed microorganisms can be utilized in their isolated andbiologically pure states, as well as being formulated into compositions.Furthermore, the disclosure provides microbial ensembles, containing atleast two members of the disclosed microorganisms, as well as methods ofutilizing said ensembles. Furthermore, the disclosure provides formethods of modulating the fowl microbiome.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is ASBI_003_05US_ST25.txt. The text file is ˜166kb, was created on Jul. 20, 2018, and is being submitted electronicallyvia EFS-Web.

BACKGROUND

The global population is predicted to increase to over 9 billion peopleby the year 2050 with a concurrent reduction in the quantity of land,water, and other natural resources available per capita. Projectionsindicate that the average domestic income will also increase, with theprojected rise in the GDP of China and India. The desire for a dietricher in animal-source proteins rises in tandem with increasing income,thus the global livestock sector will be charged with the challenge ofproducing more animal products using fewer resources. The Food andAgriculture Organization of the United Nations predict that 70% morefood will have to be produced, yet the area of arable land availablewill decrease. It is clear that the food output per unit of resourceinput will have to increase considerably in order to support the rise inpopulation.

Over recent decades the farm industry has seen fast growth in the meatsector, which has been underpinned by rising demand for poultry meat,which has consistently increased at about three times the rate ofpopulation growth over each of the past five decades.

Poultry meat, eggs, and components thereof are predominantly utilized inthe preparation of foodstuffs in many different forms. There have beenmany strategies to improve poultry and egg production throughnutritional modulations, hormone treatments, changes in animalmanagement, and selective breeding; however, the need for more efficientproduction of edible poultry foodstuffs per animal is required.

Identifying compositions and methods for sustainably increasing poultryand egg production while balancing animal health and wellbeing havebecome imperative to satisfy the needs of everyday humans in anexpanding population. Increasing the worldwide production of poultry byscaling up the total number of fowl on farms would not only beeconomically infeasible for many parts of the world, but would furtherresult in negative environmental consequences as the poultry sector'sgrowth and trends towards intensification and concentration have alreadygiven rise to a number of environmental concerns, led predominantly bythe production of far more waste than can be managed by land disposal.

Population densities of poultry in large farms are often accompanied byan increased incidence of microbial pathogens that place the poultryyield at risk, and further place the ultimate consumer of the poultry atrisk in instances of zoonotic pathogens such as those of Clostridium andSalmonella. Considering the widespread occurrence of many zoonoticpathogens, it is unlikely that poultry can be completely protected fromexposure. Research has focused on investigative means of increasingresistance to colonization in poultry exposed to these pathogens.

Thus, meeting global poultry yield expectations, by simply scaling upcurrent high-input agricultural systems—utilized in most of thedeveloped world—is simply not feasible.

There is therefore an urgent need in the art for improved methods ofincreasing poultry and egg production, while also mitigating thecolonization and spread of microbial pathogens.

SUMMARY OF THE DISCLOSURE

In some embodiments, the at least two microbial strains or the at leastone microbial strain present in a composition, or microbial ensemble, ofthe disclosure exhibit an increased utility that is not exhibited whensaid strains occur alone or when said strains are present at a naturallyoccurring concentration. In some embodiments, compositions of thedisclosure, comprising at least two microbial strains as taught herein,exhibit a synergistic effect on imparting at least one improved trait inan animal. In some embodiments, the compositions of thedisclosure—comprising one or more isolated microbes as taughtherein—exhibit markedly different characteristics/properties compared totheir closest naturally occurring counterpart. That is, the compositionsof the disclosure exhibit markedly different functional and/orstructural characteristics/properties, as compared to their closestnaturally occurring counterpart. For instance, the microbes of thedisclosure are structurally different from a microbe as it naturallyexists in a fowl gastrointestinal tract, for at least the followingreasons: said microbe can be isolated and purified, such that it is notfound in the milieu of the gastrointestinal tract, said microbe can bepresent at concentrations that do not occur in the gastrointestinaltract, said microbe can be associated with acceptable carriers that donot occur in the gastrointestinal tract, said microbe can be formulatedto be shelf-stable and exist outside the gastrointestinal tract, andsaid microbe can be combined with other microbes at concentrations thatdo not exist in the gastrointestinal tract. Further, the microbes of thedisclosure are functionally different from a microbe as it naturallyexists in a gastrointestinal tract, for at least the following reasons:said microbe when applied in an isolated and purified form can lead tomodulation of the gastrointestinal microbiome, increased weight gain,increased feed utilization, decreased amounts of microbial pathogens,decreased pathogen-associated GI lesions, said microbe can be formulatedto be shelf-stable and able to exist outside the gastrointestinalenvironment, such that the microbe now has a new utility as a supplementcapable of administration to a fowl, wherein the microbe could not havesuch a utility in it's natural state in the gastrointestinal tract, asthe microbe would be unable to survive outside the gastrointestinaltract without the intervention of the hand of man to formulate themicrobe into a shelf-stable state and impart this new utility that hasthe aforementioned functional characteristics not possessed by themicrobe in it's natural state of existence in the fowl gastrointestinaltract.

In some aspects, the present disclosure is drawn to a method ofdecreasing feed conversion ratio, increasing fowl weight, and/ordecreasing pathogen-associated lesion formation in the gastrointestinaltract of fowl, the method comprising: a) administering to a fowl aneffective amount of a shelf-stable fowl supplement comprising: i) apurified microbial population of Lactobacillus bacteria comprisingbacteria with a 16S nucleic acid sequence that is at least about 97%identical to SEQ ID NO:1, and said bacterium has a MIC score of at leastabout 0.1; and ii) a shelf-stable carrier suitable for fowladministration, wherein the fowl administered the effective amount ofthe shelf-stable fowl supplement exhibits an decrease in feed conversionratio, an increase in weight, or a decrease in pathogen-associatedlesion formation in the gastrointestinal tract, as compared to a fowlnot having been administered the supplement.

In some aspects, the present disclosure is drawn to a method ofdecreasing feed conversion ratio, increasing fowl weight, or decreasingpathogen-associated lesion formation in the gastrointestinal tract offowl, the method comprising: a) administering to a fowl an effectiveamount of a shelf-stable fowl supplement comprising: i) a purifiedmicrobial population that comprises a bacterium with a 16S nucleic acidsequence, and/or a fungus with an ITS nucleic acid sequence, which is atleast about 97% identical to a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs:1-387, and said bacterium and/or fungushave a MIC score of at least about 0.2; and ii) a shelf-stable carriersuitable for fowl administration; wherein the fowl administered theeffective amount of the shelf-stable fowl supplement exhibits a decreasein feed conversion ratio, an increase in weight, and/or a decrease inpathogen-associated lesion formation in the gastrointestinal tract, ascompared to a fowl not having been administered the supplement.

In some aspects, the present disclosure is drawn to a method of treatingpoultry for necrotic enteritis, the method comprising: a) administeringto a bird an effective amount of a shelf-stable poultry supplementcomprising: i) a purified microbial population that comprises abacterium with a 16S nucleic acid sequence, and/or a fungus with an ITSnucleic acid sequence, which is at least about 97% identical to anucleic acid sequence selected from the group consisting of SEQ IDNOs:1-387, and said bacterium and/or fungus have a MIC score of at leastabout 0.2; and ii) a shelf-stable carrier suitable for poultryadministration, wherein the bird administered the effective amount ofthe shelf-stable poultry supplement exhibits a decrease in the number ofnecrotic enteritis-causing bacteria in the gastrointestinal tract, ascompared to a bird not having been administered the supplement.

In some aspects, the present disclosure is drawn to a method of treatingpoultry for necrotic enteritis, the method comprising: administering toa bird an effective amount of a shelf-stable poultry supplementcomprising: i) a purified microbial population of Lactobacillus bacteriacomprising bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:1, and said bacterium has a MIC scoreof at least about 0.2; and ii) a shelf-stable carrier suitable forpoultry administration, wherein the poultry administered the effectiveamount of the shelf-stable poultry supplement exhibits a decrease in thenumber of necrotic enteritis-causing bacteria in the gastrointestinaltract, as compared to a bird not having been administered thesupplement.

In some aspects, the present disclosure is drawn to a method ofdecreasing feed conversion ratio, increasing fowl weight, and/ordecreasing pathogen-associated lesion formation in the gastrointestinaltract of fowl, the method comprising: a) administering to a fowl aneffective amount of a shelf-stable fowl supplement comprising: i) apurified microbial population of Lactobacillus bacteria comprisingbacteria with a 16S nucleic acid sequence that is at least about 97%identical to SEQ ID NO:374, and said bacterium has a MIC score of atleast about 0.2; and ii) a shelf-stable carrier suitable for fowladministration, wherein the fowl administered the effective amount ofthe shelf-stable fowl supplement exhibits an decrease in feed conversionratio, an increase in weight, or a decrease in pathogen-associatedlesion formation in the gastrointestinal tract, as compared to a fowlnot having been administered the supplement.

In some aspects, the present disclosure is drawn to a method ofdecreasing feed conversion ratio, increasing fowl weight, and/ordecreasing pathogen-associated lesion formation in the gastrointestinaltract of fowl, the method comprising: a) administering to a fowl aneffective amount of a shelf-stable fowl supplement comprising: i) apurified microbial population of Lactobacillus bacteria comprisingbacteria with a 16S nucleic acid sequence that is at least about 97%identical to SEQ ID NO:382, and said bacterium has a MIC score of atleast about 0.2; and ii) a shelf-stable carrier suitable for fowladministration, wherein the fowl administered the effective amount ofthe shelf-stable fowl supplement exhibits an decrease in feed conversionratio, an increase in weight, or a decrease in pathogen-associatedlesion formation in the gastrointestinal tract, as compared to a fowlnot having been administered the supplement.

In aspects, the aforementioned microbial species—that is, a purifiedmicrobial population that comprises a bacteria with a 16S nucleic acidsequence, and/or a fungi with an ITS nucleic acid sequence, which is atleast about 97% identical to a nucleic acid sequence selected from thegroup consisting of: SEQ ID NOs: 1-387—are members of a Markush group,as the present disclosure illustrates that the members belong to a classof microbes characterized by various physical and functional attributes,which can include any of the following: a) the ability to convert acarbon source into a volatile fatty acid such as acetate, butyrate,propionate, or combinations thereof; b) the ability to degrade a solubleor insoluble carbon source; c) the ability to impart an increase inweight gain to fowl administered the microbe(s); d) the ability tomodulate the microbiome of the gastrointestinal tract of fowladministered the microbe; e) the ability to be formulated into ashelf-stable composition; f) the ability to exhibit a decrease in feedconversion ratio in fowl having been administered the microbe(s); g) theability to impart a decrease in pathogen-associated lesion formation inthe gastrointestinal tract; h) the ability to impart a decrease inpathogenic microbes in the gastrointestinal tract; and/or i) possessinga MIC score of at least about 0.2 if a bacteria and possessing a MICscore of at least about 0.2 if a fungi. Thus, the members of the Markushgroup possess at least one property in common, which can be responsiblefor their function in the claimed relationship.

In some aspects, the fowl is a broiler. In some aspects, the fowlsupplement is stable under ambient conditions for at least one week. Insome aspects, the fowl supplement is formulated as an: encapsulation,tablet, capsule, pill, feed additive, food ingredient, food additive,food preparation, food supplement, water additive, water-mixed additive,heat-stabilized additive, moisture-stabilized additive, consumablesolution, consumable spray additive, consumable solid, consumable gel,injection, suppository, drench, or combinations thereof.

In some aspects, administration comprises feeding the fowl supplement toa fowl or spraying the fowl supplement onto a fowl. In some aspects, thepurified microbial population is present in the fowl supplement at aconcentration of at least 10² cells. In some aspects, the purifiedmicrobial population comprises a bacterium with a 16S nucleic acidsequence that is at least about 97% identical to a nucleic acid sequenceselected from the group consisting of: SEQ ID NOs:1-50 and 59-387. Insome aspects, the purified microbial population comprises a fungus withan ITS nucleic acid sequence that is at least about 97% identical to anucleic acid sequence selected from the group consisting of: SEQ IDNOs:51-58. In some aspects, the purified microbial population comprisesa bacterium with a 16S nucleic acid sequence that is at least about 99%identical to a nucleic acid sequence selected from the group consistingof: SEQ ID NOs:1-50 and 59-387. In some aspects, the purified microbialpopulation comprises a fungus with an ITS nucleic acid sequence that isat least about 99% identical to a nucleic acid sequence selected fromthe group consisting of: SEQ ID NOs:51-58. In some aspects, the purifiedmicrobial population comprises a bacterium with a 16S nucleic acidsequence selected from the group consisting of: SEQ ID NOs:1-50 and59-387. In some aspects, the purified microbial population comprises afungus with an ITS nucleic acid sequence selected from the groupconsisting of: SEQ ID NOs:51-58.

In some aspects, the purified microbial population comprises a bacteriumwith a 16S nucleic acid sequence and a fungus with an ITS nucleic acidsequence that is at least about 97% identical to a nucleic acid sequenceselected from the group consisting of: SEQ ID NOs:1-387. In someaspects, the purified microbial population comprises a bacteria with a16S nucleic acid sequence that is at least about 97% identical to SEQ IDNO:1. In some aspects, the purified microbial population comprises abacterium with a 16S nucleic acid sequence comprising SEQ ID NO:1, andwherein the bacterium is as deposited as PATENT201703004.

In some aspects, the purified microbial population only containsorganisms that are members of a group selected from: Lactobacillus,Clostridium, Faecalibacter, Hydrogenoanaerobacterium, Acrocarpospora,Bacillus, Subdoligranulum, Leuconostoc, Lachnospiracea, Anaerofilum,Microbacterium, Verrucosispora, Anaerofilum, Blautia, Pseudomonas,Sporobacter, Corynebacterium, Streptococcus, Paracoccus,Cellulosilyticum, Ruminococcus, Rosebura, Bacteroides, Filobasidium,Gibberella, Alatospora, Pichia, and Candida.

In some aspects, the fowl administered the effective amount of the fowlsupplement exhibits at least a 1% decrease in feed conversion ratio, atleast a 1% increase in weight, and/or at least a 1% decrease inpathogen-associated lesion formation in the gastrointestinal tract. Insome aspects, the fowl administered the effective amount of the fowlsupplement exhibits at least a 10% decrease in feed conversion ratio, atleast a 10% increase in weight, and/or at least a 10% decrease inpathogen-associated lesion formation in the gastrointestinal tract.

A shelf-stable fowl supplement capable of decreasing feed conversionratio, increasing fowl weight, or decreasing pathogen-associated lesionformation in the gastrointestinal tract of fowl, comprising: a) apurified population that comprises a bacterium with a 16S nucleic acidsequence and/or a fungus with an ITS nucleic acid sequence, which is atleast about 97% identical to a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs:1-387; and b) a shelf-stable carriersuitable for fowl administration, wherein the purified population ofbacteria and/or fungi of a) is present in the supplement in an amounteffective to decrease feed conversion ratio, increase fowl weight,and/or decrease pathogen-associated lesion formation in thegastrointestinal tract of fowl, as compared to a fowl not having beenadministered the supplement. In some aspects, the fowl administered thesupplement exhibits a decrease in feed conversion ratio as compared tofowl not having been administered the supplement. In some aspects, fowladministered the supplement exhibits a decrease in feed conversion ratioas compared to fowl not having been administered the supplement.

In some aspects, the purified population of bacteria and/or fungicomprises bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:1. In some aspects, the purifiedpopulation of bacteria and/or fungi comprises bacteria with a 16Snucleic acid sequence that is at least about 99% identical to SEQ IDNO:1. In some aspects, the purified population of bacteria and/or fungicomprises bacteria with a 16S nucleic acid sequence comprising SEQ IDNO:1. In some aspects, the purified population of bacteria and/or fungicomprises bacteria with a 16S nucleic acid sequence comprising SEQ IDNO:1, and wherein the bacteria are as deposited as PATENT201703004.

In some aspects, the shelf-stable fowl supplement further comprises: (i)a purified population of bacteria comprising a 16S nucleic acid sequencethat is at least about 97% identical to a nucleic acid sequence selectedfrom the group consisting of: SEQ ID NO:1-50 and 59-387, and/or (ii) apurified population of fungi that comprise fungi with an ITS nucleicacid sequence that is at least about 97% identical to a nucleic acidsequence selected from the group consisting of: SEQ ID NO:51-58.

In some aspects, the purified population of bacteria comprises bacteriawith a 16S nucleic acid sequence that is at least about 99% identical toa nucleic acid sequence selected from the group consisting of: SEQ IDNO:1-50 and 59-387. In some aspects, the purified population of fungicomprises fungi with an ITS nucleic acid sequence that is at least about99% identical to a nucleic acid sequence selected from the groupconsisting of: SEQ ID NO:51-58. In some aspects, the purified populationof bacteria comprises bacteria with a 16S nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:1-50 and 59-387. In some aspects,the purified population of fungi comprises fungi with an ITS nucleicacid sequence selected from the group consisting of: SEQ ID NO:51-58. Insome aspects, the purified population of bacteria comprises bacteriawith a 16S nucleic acid sequence that is at least about 97% identical toSEQ ID NO:3. In some aspects, the purified population of bacteriacomprises bacteria with a 16S nucleic acid sequence that is at leastabout 99% identical to SEQ ID NO:3. In some aspects, the purifiedpopulation of bacteria comprises bacteria with a 16S nucleic acidsequence comprising SEQ ID NO:3. In some aspects, the purifiedpopulation of bacteria comprises SEQ ID NO:1, and wherein the bacteriaare as deposited as PATENT201703001. In some aspects, both a purifiedpopulation of bacteria (i) and a purified population of fungi (ii) arepresent in the supplement.

In some aspects, the fowl supplement is formulated for administration toa broiler. In some aspects, the supplement is stable under ambientconditions for at least one week. In some aspects, the supplementformulated as an: encapsulation, encapsulation, tablet, capsule, pill,feed additive, food ingredient, food additive, food preparation, foodsupplement, water additive, water-mixed additive, heat-stabilizedadditive, moisture-stabilized additive, consumable solution, consumablespray additive, consumable solid, consumable gel, injection,suppository, drench, or combinations thereof.

In some aspects, the purified population of bacteria and/or fungi ispresent in the fowl supplement at a concentration of at least 10² cells.In some aspects, the fowl administered the supplement exhibits anincrease in weight as compared to fowl not having been administered thesupplement. In some aspects, the fowl administered the supplementexhibits a decrease in pathogen-associated lesion formation in thegastrointestinal tract as compared to fowl not having been administeredthe supplement.

In some aspects, the fowl administered the supplement exhibits adecreased incidence of Clostridium perfringens-associated lesionformation in the gastrointestinal tract as compared to fowl not havingbeen administered the supplement. In some aspects, the fowl administeredthe supplement exhibits a 1% decreased incidence of Clostridiumperfringens-associated lesion formation in the gastrointestinal tract ascompared to fowl not having been administered the supplement. In someaspects, the fowl administered the supplement exhibits a 10% decreasedincidence of Clostridium perfringens-associated lesion formation in thegastrointestinal tract as compared to fowl not having been administeredthe supplement. In some aspects, the fowl administered the supplementexhibits a 20% decreased incidence of Clostridium perfringens-associatedlesion formation in the gastrointestinal tract as compared to fowl nothaving been administered the supplement.

In some aspects, the shelf-stable supplement further comprises: (i) apurified population of bacteria comprising a 16S nucleic acid sequencethat is at least about 97% identical to a nucleic acid sequence selectedfrom the group consisting of: SEQ ID NO:1-50 and 59-387, and/or (ii) apurified population of fungi comprising an ITS nucleic acid sequencethat is at least about 97% identical to a nucleic acid sequence selectedfrom the group consisting of: SEQ ID NO:51-58.

In some embodiments, the present disclosure is drawn to a method fordecreasing feed conversion ratio, increasing fowl weight, or decreasingpathogen-associated lesion formation in the gastrointestinal tract offowl, the method comprising: a) administering to a fowl an effectiveamount of a shelf-stable fowl supplement comprising: i) a purifiedmicrobial population that comprises a bacterium with a 16S nucleic acidsequence at least about 97% identical to SEQ ID NOs:13, 346, 19, or 22,and said bacterium has a MIC score of at least about 0.2; and ii) ashelf-stable carrier suitable for fowl administration, wherein the fowladministered the effective amount of the shelf-stable fowl supplementexhibits a decrease in feed conversion ratio, an increase in weight,and/or a decrease in pathogen-associated lesion formation in thegastrointestinal tract, as compared to a fowl not having beenadministered the supplement.

A shelf-stable fowl supplement capable of decreasing feed conversionratio, increasing fowl weight, or decreasing pathogen-associated lesionformation in the gastrointestinal tract of fowl, comprising: a) apurified population that comprises a bacterium with a 16S nucleic acidsequence at least about 97% identical to SEQ ID NOs:13, 346, 19, or 22;and b) a shelf-stable carrier suitable for fowl administration; whereinthe purified population of bacteria of a) is present in the supplementin an amount effective to decrease feed conversion ratio, increase fowlweight, and/or decrease pathogen-associated lesion formation in thegastrointestinal tract of fowl, as compared to a fowl not having beenadministered the supplement.

A method for decreasing feed conversion ratio, increasing fowl weight,and/or decreasing pathogen-associated lesion formation in thegastrointestinal tract of fowl, the method comprising: a) administeringto a fowl an effective amount of a shelf-stable fowl supplementcomprising: i) a purified microbial population of Bacillus,Lactobacillus, or Eubacterium bacteria comprising bacteria with a 16Snucleic acid sequence selected from SEQ ID NOs:13, 346, 19, or 22, andsaid bacterium has a MIC score of at least about 0.2; and ii) ashelf-stable carrier suitable for fowl administration, wherein the fowladministered the effective amount of the shelf-stable fowl supplementexhibits an decrease in feed conversion ratio, an increase in weight, ora decrease in pathogen-associated lesion formation in thegastrointestinal tract, as compared to a fowl not having beenadministered the supplement.

A shelf-stable fowl supplement capable of decreasing feed conversionratio, increasing fowl weight, or decreasing pathogen-associated lesionformation in the gastrointestinal tract of fowl, comprising: a) apurified population of Bacillus, Lactobacillus, or Eubacterium bacteriacomprising bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NOs:13, 346, 19, or 22; and b) ashelf-stable carrier suitable for fowl administration, wherein thepurified population of Bacillus, Lactobacillus, or Eubacterium bacteriaof a) is present in the supplement in an amount effective to decreasefeed conversion ratio, increase fowl weight, or decreasepathogen-associated lesion formation in the gastrointestinal tract offowl, as compared to a fowl not having been administered the supplement.

A method of treating poultry for necrotic enteritis, the methodcomprising: a) administering to a bird an effective amount of ashelf-stable poultry supplement comprising: i) a purified microbialpopulation that comprises a bacterium with a 16S nucleic acid sequenceat least about 97% identical to a nucleic acid sequence selected fromSEQ ID NOs:13, 346, 19, or 22, and said bacterium has a MIC score of atleast about 0.2; and ii) a shelf-stable carrier suitable for poultryadministration, wherein the bird administered the effective amount ofthe shelf-stable poultry supplement exhibits a decrease in the number ofnecrotic enteritis-causing bacteria in the gastrointestinal tract, ascompared to a bird not having been administered the supplement.

A method of treating poultry for necrotic enteritis, the methodcomprising: a) administering to a bird an effective amount of ashelf-stable poultry supplement comprising: i) a purified microbialpopulation of Bacillus, Lactobacillus, or Eubacterium bacteriacomprising bacteria with a 16S nucleic acid sequence selected from SEQID NOs:13, 346, 19, or 22, and said bacterium has a MIC score of atleast about 0.2; and ii) a shelf-stable carrier suitable for poultryadministration, wherein the poultry administered the effective amount ofthe shelf-stable poultry supplement exhibits a decrease in the number ofnecrotic enteritis-causing bacteria in the gastrointestinal tract, ascompared to a bird not having been administered the supplement.

In aspects, the aforementioned microbial species—that is, a purifiedmicrobial population that comprises a bacteria with a 16S nucleic acidsequence, and/or a fungi with an ITS nucleic acid sequence, which is atleast about 97% identical to a nucleic acid sequence selected from thegroup consisting of: SEQ ID NOs: 1-387—are members of a Markush group,as the present disclosure illustrates that the members belong to a classof microbes characterized by various physical and functional attributes,which can include any of the following: a) the ability to convert acarbon source into a volatile fatty acid such as acetate, butyrate,propionate, or combinations thereof; b) the ability to degrade a solubleor insoluble carbon source; c) the ability to impart an increase inweight gain to fowl administered the microbe(s); d) the ability tomodulate the microbiome of the gastrointestinal tract of fowladministered the microbe; e) the ability to be formulated into ashelf-stable composition; f) the ability to exhibit a decrease in feedconversion ratio in fowl having been administered the microbe(s); g) theability to impart a decrease in pathogen-associated lesion formation inthe gastrointestinal tract; h) the ability to impart a decrease inpathogenic microbes in the gastrointestinal tract; and/or i) possessinga MIC score of at least about 0.2 if a bacteria and possessing a MICscore of at least about 0.2 if a fungi. Thus, the members of the Markushgroup possess at least one property in common, which can be responsiblefor their function in the claimed relationship.

Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedures

Some microorganisms described in this application were deposited withthe United States Department of Agriculture (USDA) Agricultural ResearchService (ARS) Culture Collection) (NRRL®), located at 1815 N. UniversitySt., Peoria, Ill. 61604, USA. Some microorganisms described in thisapplication were deposited with the Bigelow National Center for MarineAlgae and Microbiota, located at 60 Bigelow Drive, East Boothbay, Maine04544, USA. Some microorganisms described in this application weredeposited with the American Type Culture Collection (ATCC), located at10801 University Boulevard, Manassas, Va. 20108, USA.

The deposits were made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. The NRRL®, ATCC, and Bigelow NationalCenter for Marine Algae and Microbiota accession numbers for theaforementioned Budapest Treaty deposits are provided in Table 3. Theaccession numbers and corresponding dates of deposit for themicroorganisms described in this application are separately provided inTable 45.

The strains designated in the below table have been deposited in thelabs of Ascus Biosciences, Inc. since at least Mar. 1, 2016. Strainscomprising SEQ ID NOs: 386 and 387 were isolated prior to, and storedsince Summer 2017.

In Table 1, the closest predicted hits for taxonomy of the microbes arelisted in columns 2 and 5. Column 2 is the top taxonomic hit predicatedby BLAST, and column 5 is the top taxonomic hit for genus+speciespredicted by BLAST. The strains designated in the below table have beendeposited in the labs of Ascus Biosciences, Inc. since at least Mar. 1,2016.

Table 1 lists strain designations of the bacteria and fungi of thepresent disclosure. If a letter in parentheses follows any of the straindesignations, then that indicates that each of those strains havevariants that share at least 97% sequence identity with the referencestrain with the (A) parenthetical. Ascusbbr_5796 (A) has two variants,Ascusbbr_5796(B) and Ascusbbr_5796(C) that share 97.8% and 98.2%sequence identity, respectively, with Ascusbbr_5796 (A).Ascusbbr_14690(A) has two variants, Ascusbbr_14690(B) andAscusbbr_14690(C) that share 97.8% and 98.2% sequence identity,respectively, with Ascusbbr_14690(A). Ascusbbr_38717(A) shares 98.6%sequence identity with Ascusbbr_38717(B). Ascusbbr_33(A) shares 98.2%sequence identity with Ascusbbr_33(B). Ascusbbr_409(B), Ascusbbr_409(C),Ascusbbr_409(D), share 98.2%, 97.3%, and 97.8% sequence identity,respectively, with Ascusbbr_409(B). Ascus_331885(B) and Ascus_331885(C)share 97.8% and 97.3% sequence identity, respectively, withAscus_331885(A). Ascusbbr_247(A) shares 97.8% sequence identity withAscusbbr_247(B). Ascusbbr_10593(A) shares 99.6% sequence identity withAscusbbr_10593(B). Ascusbbr_32731(A) shares 97.3% sequence identity withAscusbbr_32731(B). Ascusbbr_1436(A) shares 97.8% sequence identity withAscusbbr_1436(B). Ascusbbr_265(A) shares 99.6% sequence identity withAscusbbr_265(B).

TABLE 1 Microbes of the present disclosure, including bacteria (1-99)and fungi (100-107). Predicted Closest Taxa BLAST Taxonomic of IsolatedBLAST Taxonomic BLAST % Query Top Hit w/ Genus + Microbes Top Hit Ident.Cover Species 1. Lactobacillus Lactobacillus 98% 100% Lactobacillus(Genus) crispatus crispatus 2. Lachnospiraceae Bacterium ic1296 98% 91%Ruminococcus Clostridium gnavus Cluster XIVa (Family + Cluster) 3.Lactobacillus Lactobacillus 100% 100% Lactobacillus (Genus) crispatuscrispatus 4. Lactobacillus Lactobacillus 100% 100% Lactobacillus (Genus)crispatus crispatus 5. Lactobacillus Lactobacillus 100% 100%Lactobacillus (Genus) crispatus crispatus 6. Lactobacillus Lactobacillus99% 100% Lactobacillus (Genus) vaginalis vaginalis 7. LactobacillusLactobacillus 99% 100% Lactobacillus (Genus) vaginalis vaginalis 8.Lactobacillus Lactobacillus 99% 98% Lactobacillus (Genus) vaginalisvaginalis 9. Lactobacillus Lactobacillus 99% 100% Lactobacillus (Genus)johnsonii johnsonii 10. Faecalibacterium Faecalibacterium 90% 98%Faecalibacterium (Genus) sp. prausnitzii 11. Lactobacillus Lactobacillus99% 100% Lactobacillus (Genus) johnsonii johnsonii 12. HydrogenoanaeroClostridium sp. 91% 98% Butyrivibrio bacterium hungatei (Genus) 13.Peptostrepto Clostridium sp. 92% 100% [Eubacterium] coccaceae tenueClostridium Cluster XI (Family + Cluster) 14. Acrocarpospora Nonomuraeasp. 99% 88% Microbispora (Genus) rosea 15. Lactobacillus Lactobacillus99% 98% Lactobacillus (Genus) helveticus helveticus 16. Bacillus (Genus)Bacillus subtilis 99% 100% Bacillus subtilis 17. Bacillus (Genus)Bacillus subtilis 99% 100% Bacillus subtilis 18. LactobacillusLactobacillus 99% 95% Lactobacillus (Genus) coleohominis coleohominis19. Subdoligranulum Bacterium ic1340 90% 98% Anaerofilum (Genus)pentosovorans 20. Subdoligranulum Firmicutes 99% 100% Faecalibacterium(Genus) bacterium prausnitzii 21. Lachnospiraceae Clostridium sp. 95%100% Eubacterium Clostridium fissicatena Cluster XIVa (Family + Cluster)22. Lactobacillus Lactobacillus 99% 95% Lactobacillus (Genus)coleohominis coleohominis 23. Lactobacillus Lactobacillus reuteri 100%100% Lactobacillus (Genus) reuteri 24. Lactobacillus Lactobacillusreuteri 100% 100% Lactobacillus (Genus) reuteri 25. LactobacillusLactobacillus reuteri 100% 100% Lactobacillus (Genus) reuteri 26.Lactobacillus Lactobacillus reuteri 100% 100% Lactobacillus (Genus)reuteri 27. Leuconostoc Leuconostoc 99% 100% Leuconostoc (Genus)mesenteroides mesenteroides 28. Lachnospiracea Lachnospiraceae 96% 100%Eubacterium hallii incertae sedis bacterium (Genus) 29. LactobacillusLactobacillus reuteri 99% 100% Lactobacillus (Genus) reuteri 30.Lactobacillus Lactobacillus reuteri 99% 100% Lactobacillus (Genus)reuteri 31. Lactobacillus Lactobacillus reuteri 99% 100% Lactobacillus(Genus) reuteri 32. Anaerofilum Clostridiales 88% 97% Ruthenibacterium(Genus) bacterium lactatiformans 33. Lachnospiracea Blautia 96% 100%Blautia incertae sedis hydrogenotrophica hydrogenotrophica (Genus) 34.Lachnospiraceae Clostridium 98% 100% Clostridium Clostridiumsaccharolyticum-like clostridioforme Cluster XIVa K10 (Family + Cluster)35. Lachnospiraceae Clostridium 98% 100% Clostridium Clostridiumsaccharolyticum-like clostridioforme Cluster XIVa K10 (Family + Cluster)36. Microbacterium Pseudoclavibacter sp. 95% 99% Pseudoclavibacter(Genus) caeni 37. Verrucosispora Verrucosispora sp. 99% 100%Verrucosispora (Genus) wenchangensis 38. Anaerofilum Faecalibacterium93% 100% Faecalibacterium (Genus) prausnitzii prausnitzii 39.Clostridium sensu Candidatus 90% 91% Peptoclostridium stricto (Genus)Arthromitus sp difficile 40. Lactobacillus Lactobacillus 96% 100%Lactobacillus (Genus) helveticus helveticus 41. LactobacillusLactobacillus 99% 100% Lactobacillus (Genus) salivarius salivarius 42.Lactobacillus Lactobacillus 99% 100% Lactobacillus (Genus) salivariussalivarius 43. Lachnospiraceae Roseburia 93% 100% Roseburia Clostridiuminulinivorans inulinivorans Cluster XIVa (Family + Cluster) 44. Blautia(Genus) Ruminococcus obeum 94% 100% Ruminococcus obeum 45. LactobacillusLactobacillus oris 99% 99% Lactobacillus oris (Genus) 46. LactobacillusLactobacillus oris 99% 99% Lactobacillus oris (Genus) 47. LactobacillusLactobacillus 99% 83% Lactobacillus (Genus) crispatus crispatus 48.Lactobacillus Lactobacillus reuteri 94% 88% Lactobacillus (Genus)reuteri 49. Pseudomonas Pseudomonas 99% 100% Pseudomonas (Genus)chengduensis chengduensis 50. Sporobacter Syntrophomonadaceae 86% 82%Clostridium (Genus) bacterium sphenoides 51. Lactobacillus Lactobacillus99% 83% Lactobacillus (Genus) crispatus crispatus 52. LactobacillusLactobacillus 98% 82% Lactobacillus (Genus) salivarius salivarius 53.Lactobacillus Lactobacillus 98% 82% Lactobacillus (Genus) helveticushelveticus 54. Lachnospiracea Blautia 96% 100% Blautia incertae sedishydrogenotrophica hydrogenotrophica (Genus) 55. LactobacillusLactobacillus 97% 83% Lactobacillus (Genus) crispatus crispatus 56.Lactobacillus Lactobacillus 97% 100% Lactobacillus (Genus) vaginalisvaginalis 57. Peptostrepto Clostridium sp. 93% 100% Romboutsia coccaceaelituseburensis Clostridium Cluster XI (Family + Cluster) 58.Lactobacillus Lactobacillus 96% 100% Lactobacillus (Genus) crispatuscrispatus 59. Lactobacillus Lactobacillus 98% 94% Lactobacillus (Genus)johnsonii johnsonii 60. Lactobacillus Lactobacillus reuteri 96% 100%Lactobacillus (Genus) reuteri 61. Lactobacillus Lactobacillus 93% 83%Lactobacillus (Genus) crispatus crispatus 62. CorynebacteriumCorynebacterium 100% 100% Corynebacterium (Genus) glutamicum glutamicum63. Streptococcus Streptococcus 97% 94% Streptococcus (Genus)hyovaginalis hyovaginalis 64. Lactobacillus Lactobacillus aviarius 99%100% Lactobacillus (Genus) aviarius 65. Lactobacillus Lactobacillusaviarius 99% 100% Lactobacillus (Genus) aviarius 66. LactobacillusLactobacillus aviarius 99% 100% Lactobacillus (Genus) aviarius 67.Corynebacterium Corynebacterium 100% 100% Corynebacterium (Genus)xerosis xerosis 68. Peptostrepto Romboutsia 98% 100% Romboutsiacoccaceae lituseburensis lituseburensis (Clostridium Cluster XI)(Family + Cluster) 69. Corynebacterium Corynebacterium 100% 100%Corynebacterium (Genus) falsenii falsenii 70. CorynebacteriumCorynebacterium 98% 97% Corynebacterium (Genus) ammoniagenesammoniagenes 71. Lachnospiraceae Desulfotomaculum sp. 96% 100%Clostridium (Clostridium sphenoides Cluster XIVa) (Family + Cluster) 72.Lachnospiraceae Desulfotomaculum sp. 96% 100% Clostridium (Clostridiumsphenoides Cluster XIVa) (Family + Cluster) 73. LachnospiraceaEubacterium sp. 98% 100% Eubacterium incertae sedis fissicatena (Genus)74. Lachnospiracea Eubacterium sp. 98% 100% Eubacterium incertae sedisfissicatena (Genus) 75. Ruminococcaceae Bacterium 89% 78%Ruminiclostridium (Clostridium thermocellum Cluster III) (Family +Cluster) 76. Lactobacillus Lactobacillus 100% 100% Lactobacillus (Genus)pentosus pentosus 77. Streptococcus Swine fecal bacterium 100% 100%Streptococcus (Genus) alactolyticus 78. Lachnospiraceae Lachnospiraceae91% 92% Blautia producta (Clostridium bacterium Cluster XIVa) (Family +Cluster) 79. Lactobacillus Lactobacillus 100% 100% Lactobacillus (Genus)helveticus helveticus 80. Lactobacillus Lactobacillus 100% 100%Lactobacillus (Genus) helveticus helveticus 81. Paracoccus Paracoccus99% 99% Paracoccus (Genus) alcaliphilus alcaliphilus 82.Cellulosilyticum Ruminococcus sp. 97% 100% Hydrogenoan (Genus)aerobacterium saccharovorans 83. Blautia (Genus) Blautia glucerasea 89%100% Blautia glucerasea 84. Corynebacterium Corynebacterium 99% 100%Corynebacterium (Genus) flavescens flavescens 85. LachnospiraceaEubacteriaceae 98% 92% Coprococcus catus incertae sedis bacterium(Genus) 86. Corynebacterium Corynebacterium 100% 100% Corynebacterium(Genus) callunae callunae 87. Corynebacterium Corynebacterium 99% 100%Corynebacterium (Genus) stationis stationis 88. Ruminococcus Clostridiumsp. 92% 96% Blautia producta (Genus) 89. Lactobacillus Lactobacillus 92%100% Lactobacillus (Genus) intestinalis intestinalis 90. RoseburiaBacterium AC2012 92% 99% Frisingicoccus (Genus) caecimuris 91.Lachnospiraceae Clostridium sp. 90% 99% Eubacterium (Clostridiumventriosum Cluster XIVa) (Family + Cluster) 92. LachnospiraceaeClostridiales 99% 99% Clostridium (Clostridium bacteriumlactatifermentans Cluster XIVb) (Family + Cluster) 93. Clostridium sensuClostridium sp. 99% 100% Clostridium stricto (Genus) thermobutyricum 94.Bacteroides dorei Bacteroides dorei 100% 100% Bacteroides dorei 95.Lactobacillus Lactobacillus reuteri 95% 100% Lactobacillus (Genus)reuteri 96. Lactobacillus Lactobacillus reuteri 96% 98% Lactobacillus(Genus) reuteri 97. Lactobacillus Lactobacillus 100% 100% Lactobacillus(Genus) saerimneri saerimneri 98. Clostridium Clostridium 99% 100%Clostridium (Genus) beijerinckii beijerinckii 99. ClostridiumClostridium 100% 99% Clostridium (Genus) saccharolyticum saccharolyticum100. Nectriaceae Fusarium annulatum 100% 100% Fusarium (Family)annulatum 101. Filobasidium Uncultured fungus 100% 100% Cryptococcusfloriforme magnus (Genus + species) 102. Gibberella zeae Fusariumasiaticum 100% 100% Fusarium (Genus + species) asiaricum 103. AlatosporaUncultured 83% 81% Gymnoascus reesii (Genus) Gymnoascus 104.Hypocreaceae Geotrichum sp. 100% 100% Geotrichum (Family) candidum 105.Pichia Pichia fermentans 100% 100% Pichia fermentans fermentans (Genus +species) 106. Candida Candida railenensis 99% 100% Candida railenensisrailenensis (Genus + species) 107. Hypocreaceae Uncultured fungus 100%100% Geotrichum (Family) candidum Sequence Predicted Identifier ClosestTaxa for of Isolated BLAST % Query Associated MIC Microbes Ident. CoverStrain Designation Marker Score 1. Lactobacillus 98% 100% Ascusbbr_4729SEQ ID 0.76676 (Genus) NO: 1 2. Lachnospiraceae 95% 98% Ascusbbr_339 SEQID 0.62924 Clostridium NO: 2 Cluster XIVa (Family + Cluster) 3.Lactobacillus 100% 100% Ascusbbr_5796(A) SEQ ID 0.61325 (Genus) NO: 3 4.Lactobacillus 100% 100% Ascusbbr_5796(B) SEQ ID 0.61325 (Genus) NO: 3695. Lactobacillus 100% 100% Ascusbbr_5796(C) SEQ ID 0.61325 (Genus) NO:370 6. Lactobacillus 99% 100% Ascusbbr_38717(A) SEQ ID 0.59229 (Genus)NO: 4 7. Lactobacillus 99% 100% Ascusbbr_38717(B) SEQ ID 0.59229 (Genus)NO: 373 8. Lactobacillus 99% 98% Ascusbbr_170211 SEQ ID 0.58403 (Genus)NO: 5 9. Lactobacillus 99% 100% Ascusbbr_1686 SEQ ID 0.57845 (Genus) NO:6 10. Faecalibacterium 89% 97% Ascusbbr_1789 SEQ ID 0.56099 (Genus) NO:7 11. Lactobacillus 99% 100% Ascusbbr_3820 SEQ ID 0.55862 (Genus) NO: 812. Hydrogenoanaero 86% 82% Ascusbbr_173 SEQ ID 0.55675 bacterium NO: 9(Genus) 13. Peptostrepto 91% 98% Ascusbbr_3089 SEQ ID 0.55548 coccaceaeNO: 10 Clostridium Cluster XI (Family + Cluster) 14. Acrocarpospora 95%100% Ascusbbr_167 SEQ ID 0.5442 (Genus) NO: 11 15. Lactobacillus 99% 98%Ascusbbr_301568 SEQ ID 0.53873 (Genus) NO: 12 16. Bacillus (Genus) 99%100% Ascusbbr_33(A) SEQ ID 0.53686 NO: 13 17. Bacillus (Genus) 99% 100%Ascusbbr_33(B) SEQ ID 0.53686 NO: 374 18. Lactobacillus 99% 95%Ascusbbr_25200 SEQ ID 0.52435 (Genus) NO: 14 19. Subdoligranulum 88% 91%Ascusbbr_84 SEQ ID 0.52174 (Genus) NO: 15 20. Subdoligranulum 96% 100%Ascusbbr_136 SEQ ID 0.51373 (Genus) NO: 16 21. Lachnospiraceae 94% 98%Ascusbbr_128 SEQ ID 0.51348 Clostridium NO: 17 Cluster XIVa (Family +Cluster) 22. Lactobacillus 99% 95% Ascusbbr_322104 SEQ ID 0.50724(Genus) NO: 18 23. Lactobacillus 100% 100% Ascusbbr_409(A) SEQ ID0.50572 (Genus) NO: 19 24. Lactobacillus 100% 100% Ascusbbr_409(B) SEQID 0.50572 (Genus) NO: 375 25. Lactobacillus 100% 100% Ascusbbr_409(C)SEQ ID 0.50572 (Genus) NO: 376 26. Lactobacillus 100% 100%Ascusbbr_409(D) SEQ ID 0.50572 (Genus) NO: 377 27. Leuconostoc 99% 100%Ascusbbr_127 SEQ ID 0.4955 (Genus) NO: 20 28. Lachnospiracea 91% 100%Ascusbbr_14834 SEQ ID 0.49531 incertae sedis NO: 21 (Genus) 29.Lactobacillus 99% 100% Ascusbbr_331885(A) SEQ ID 0.49378 (Genus) NO: 2230. Lactobacillus 99% 100% Ascusbbr_331885(B) SEQ ID 0.49378 (Genus) NO:378 31. Lactobacillus 99% 100% Ascusbbr_331885(C) SEQ ID 0.49378 (Genus)NO: 379 32. Anaerofilum 88% 98% Ascusbbr_31 SEQ ID 0.48633 (Genus) NO:23 33. Lachnospiracea 96% 100% Ascusbbr_2307 SEQ ID 0.48546 incertaesedis NO: 24 (Genus) 34. Lachnospiraceae 91% 100% Ascusbbr_247(A) SEQ ID0.48546 Clostridium NO: 25 Cluster XIVa (Family + Cluster) 35.Lachnospiraceae 91% 100% Ascusbbr_247(B) SEQ ID 0.48546 Clostridium NO:380 Cluster XIVa (Family + Cluster) 36. Microbacterium 99% 79%Ascusbbr_19 SEQ ID 0.47772 (Genus) NO: 26 37. Verrucosispora 97% 98%Ascusbbr_69 SEQ ID 0.47757 (Genus) NO: 27 38. Anaerofilum 93% 100%Ascusbbr_94 SEQ ID 0.46645 (Genus) NO: 28 39. Clostridium sensu 89% 91%Ascusbbr_313454 SEQ ID 0.46594 stricto (Genus) NO: 29 40. Lactobacillus96% 100% Ascusbbr_351000 SEQ ID 0.46296 (Genus) NO: 30 41. Lactobacillus99% 100% Ascusbbr_1436(A) SEQ ID 0.46076 (Genus) NO: 31 42.Lactobacillus 99% 100% Ascusbbr_1436(B) SEQ ID 0.46076 (Genus) NO: 38343. Lachnospiraceae 93% 100% Ascusbbr_28 SEQ ID 0.46028 Clostridium NO:32 Cluster XIVa (Family + Cluster) 44. Blautia (Genus) 94% 100%Ascusbbr_144 SEQ ID 0.45742 NO: 33 45. Lactobacillus 99% 99%Ascusbbr_42760(A) SEQ ID 0.43682 (Genus) NO: 34 46. Lactobacillus 99%99% Ascusbbr_42760(B) SEQ ID 0.43682 (Genus) NO: 384 47. Lactobacillus99% 83% Ascusbbr_134994 SEQ ID 0.434 (Genus) NO: 35 48. Lactobacillus94% 88% Ascusbbr_358773 SEQ ID 0.43348 (Genus) NO: 36 49. Pseudomonas99% 100% Ascusbbr_2503 SEQ ID 0.42622 (Genus) NO: 37 50. Sporobacter 84%82% Ascusbbr_312 SEQ ID 0.42622 (Genus) NO: 39 51. Lactobacillus 99% 83%Ascusbbr_140914 SEQ ID 0.40935 (Genus) NO: 40 52. Lactobacillus 98% 82%Ascusbbr_257627 SEQ ID 0.40775 (Genus) NO: 41 53. Lactobacillus 98% 82%Ascusbbr_310088 SEQ ID 0.40576 (Genus) NO: 42 54. Lachnospiracea 96%100% Ascusbbr_91 SEQ ID 0.40345 incertae sedis NO: 43 (Genus) 55.Lactobacillus 97% 83% Ascusbbr_150100 SEQ ID 0.40128 (Genus) NO: 44 56.Lactobacillus 97% 100% Ascusbbr_252028 SEQ ID 0.3998 (Genus) NO: 45 57.Peptostrepto 92% 100% Ascusbbr_2158 SEQ ID 0.39816 coccaceae NO: 46Clostridium Cluster XI (Family + Cluster) 58. Lactobacillus 96% 100%Ascusbbr_373 SEQ ID 0.37614 (Genus) NO: 47 59. Lactobacillus 98% 94%Ascusbbr_1802 SEQ ID 0.37123 (Genus) NO: 48 60. Lactobacillus 96% 100%Ascusbbr_107 SEQ ID 0.37123 (Genus) NO: 49 61. Lactobacillus 93% 83%Ascusbbr_1727 SEQ ID 0.36309 (Genus) NO: 50 62. Corynebacterium 100%100% Ascusbbr_226 SEQ ID 0.75897 (Genus) NO: 338 63. Streptococcus 97%94% Ascusbbr_17 SEQ ID 0.62924 (Genus) NO: 339 64. Lactobacillus 99%100% Ascusbbr_14690(A) SEQ ID 0.60061 (Genus) NO: 340 65. Lactobacillus99% 100% Ascusbbr_14690(B) SEQ ID 0.60061 (Genus) NO: 371 66.Lactobacillus 99% 100% Ascusbbr_14690(C) SEQ ID 0.60061 (Genus) NO: 37267. Corynebacterium 100% 100% Ascusbbr_18 SEQ ID 0.58366 (Genus) NO: 34168. Peptostrepto 98% 100% Ascusbbr_7363 SEQ ID 0.57242 coccaceae NO: 342(Clostridium Cluster XI) (Family + Cluster) 69. Corynebacterium 100%100% Ascusbbr_35 SEQ ID 0.49929 (Genus) NO: 343 70. Corynebacterium 98%97% Ascusbbr_7779 SEQ ID 0.48127 (Genus) NO: 344 71. Lachnospiraceae 95%100% Ascusbbr_10593(A) SEQ ID 0.47763 (Clostridium NO: 345 Cluster XIVa)(Family + Cluster) 72. Lachnospiraceae 95% 100% Ascusbbr_10593(B) SEQ ID0.47763 (Clostridium NO: 381 Cluster XIVa) (Family + Cluster) 73.Lachnospiracea 93% 98% Ascusbbr_32731(A) SEQ ID 0.47124 incertae sedisNO: 346 (Genus) 74. Lachnospiracea 93% 98% Ascusbbr_32731(B) SEQ ID0.47124 incertae sedis NO: 382 (Genus) 75. Ruminococcaceae 86% 79%Ascusbbr_359892 SEQ ID 0.39553 (Clostridium NO: 347 Cluster III)(Family + Cluster) 76. Lactobacillus 100% 100% Ascusbbr_25721 SEQ ID0.39537 (Genus) NO: 348 77. Streptococcus 99% 100% Ascusbbr_72076 SEQ ID0.38425 (Genus) NO: 349 78. Lachnospiraceae 89% 97% Ascusbbr_6097 SEQ ID0.37484 (Clostridium NO: 350 Cluster XIVa) (Family + Cluster) 79.Lactobacillus 100% 100% Ascusbbr_265(A) SEQ ID 0.37167 (Genus) NO: 35180. Lactobacillus 100% 100% Ascusbbr_265(B) SEQ ID 0.37167 (Genus) NO:385 81. Paracoccus 99% 99% Ascusbbr_323376 SEQ ID 0.36852 (Genus) NO:352 82. Cellulosilyticum 84% 98% Ascusbbr_36257 SEQ ID 0.36078 (Genus)NO: 353 83. Blautia (Genus) 89% 100% Ascusbbr_6957 SEQ ID 0.35528 NO:354 84. Corynebacterium 99% 100% Ascusbbr_38 SEQ ID 0.35488 (Genus) NO:355 85. Lachnospiracea 87% 99% Ascusbbr_13398 SEQ ID 0.34774 incertaesedis NO: 356 (Genus) 86. Corynebacterium 100% 100% Ascusbbr_57 SEQ ID0.34405 (Genus) NO: 357 87. Corynebacterium 99% 100% Ascusbbr_285160 SEQID 0.32892 (Genus) NO: 358 88. Ruminococcus 91% 96% Ascusbbr_37385 SEQID 0.3236 (Genus) NO: 359 89. Lactobacillus 92% 100% Ascusbbr_118124 SEQID 0.31115 (Genus) NO: 360 90. Roseburia 91% 100% Ascusbbr_32592 SEQ ID0.29912 (Genus) NO: 361 91. Lachnospiraceae 92% 91% Ascusbbr_110856 SEQID 0.29418 (Clostridium NO: 362 Cluster XIVa) (Family + Cluster) 92.Lachnospiraceae 97% 99% Ascusbbr_185064 SEQ ID 0.21604 (Clostridium NO:363 Cluster XIVb) (Family + Cluster) 93. Clostridium sensu 99% 99%Ascusbbr_3315 SEQ ID 0.20534 stricto (Genus) NO: 364 94. Bacteroidesdorei 100% 100% Ascusbbr_578 SEQ ID 0.74887 NO: 365 95. Lactobacillus95% 100% Ascusbbr_21169 SEQ ID 0.47787 (Genus) NO: 366 96. Lactobacillus96% 98% Ascusbbr_110856 SEQ ID 0.39178 (Genus) NO: 367 97. Lactobacillus100% 100% Ascusbbr_830 SEQ ID 0.33782 (Genus) NO: 368 98. Clostridium99% 100% Ascusbbr_2676 SEQ ID 0.40777 (Genus) NO: 386 99. Clostridium100% 99% Ascusbbr_105932 SEQ ID 0.47763 (Genus) NO: 387 100. Nectriaceae100% 100% Ascusfbr_15 SEQ ID 0.42622 (Family) NO: 51 101. Filobasidium100% 100% Ascusfbr_131 SEQ ID 0.42622 floriforme NO: 52 (Genus +species) 102. Gibberella zeae 100% 100% Ascusfbr_26 SEQ ID 0.36913(Genus + species) NO: 53 103. Alatospora 83% 81% Ascusfbr_2616 SEQ ID0.33927 (Genus) NO: 54 104. Hypocreaceae 100% 100% Ascusfbr_12 SEQ ID0.32217 (Family) NO: 55 105. Pichia 100% 100% Ascusfbr_53 SEQ ID 0.30645fermentans NO: 56 (Genus + species) 106. Candida 99% 100% Ascusfbr_1379SEQ ID 0.28513 railenensis NO: 57 (Genus + species) 107. Hypocreaceae100% 100% Ascusfbr_122 SEQ ID 0.25801 (Family) NO: 58

In some embodiments, the isolated microbial strains of the presentdisclosure further encompass mutants thereof. In some embodiments, thepresent disclosure further contemplates microbial strains having all ofthe identifying characteristics of the presently disclosed microbialstrains.

TABLE 2 Microbial Deposits Corresponding to the Microbes of Table 1Sequence Identifier for Predicted Taxa Predicted Taxa of StrainAssociated of Isolated Isolated Microbes Designation Marker Deposit #Microbes Lactobacillus Ascusbbr_4729 SEQ ID PATENT201703004 Peptostrepto(Genus) NO: 1 coccaceae Clostridium Cluster XI (Family + Cluster)Lachnospiraceae Ascusbbr_339 SEQ ID PTA-124016, LactobacillusClostridium NO: 2 PTA-124039 (Genus) Cluster XIVa (Family + Cluster)Lactobacillus Ascusbbr_5796 SEQ ID PATENT201703001, Lactobacillus(Genus) (A) NO: 3 PATENT201703003, (Genus) PATENT201703004, B-67267Lactobacillus Ascusbbr_5796 SEQ ID PTA-124039 (Genus) (B) NO: 369Lactobacillus Ascusbbr_5796 SEQ ID PATENT201703002 (Genus) (C) NO: 370Lactobacillus Ascusbbr_38717 SEQ ID PATENT201703002, Lactobacillus(Genus) (A) NO: 4 PATENT201703003, (Genus) PATENT201703004, B-67268Lactobacillus Ascusbbr_38717 SEQ ID PATENT201703001 (Genus) (B) NO: 373Lactobacillus Ascusbbr_170211 SEQ ID PATENT201703002 Lactobacillus(Genus) NO: 5 (Genus) Lactobacillus Ascusbbr_1686 SEQ ID PTA-124016,Corynebacterium (Genus) NO: 6 PTA-124039, (Genus) PATENT201703001,PATENT201703002, PATENT201703003, PATENT201703004, B-67270Faecalibacterium Ascusbbr_1789 SEQ ID PTA-124016, Streptococcus (Genus)NO: 7 PTA-124039, (Genus) PATENT201703001 Lactobacillus Ascusbbr_3820SEQ ID PTA-124039, Lactobacillus (Genus) NO: 8 PATENT201703002 (Genus)Lactobacillus (Genus) Lactobacillus (Genus) Hydrogenoanaero Ascusbbr_173SEQ ID PTA-124016, Corynebacterium bacterium NO: 9 PTA-124039 (Genus)(Genus) Peptostreptococcaceae Ascusbbr_3089 SEQ ID PTA-124016Peptostreptococcaceae Clostridium NO: 10 (Clostridium Cluster XI ClusterXI) (Family + (Family + Cluster) Cluster) Acrocarpospora Ascusbbr_167SEQ ID Corynebacterium (Genus) NO: 11 (Genus) LactobacillusAscusbbr_301568 SEQ ID Corynebacterium (Genus) NO: 12 (Genus) BacillusAscusbbr_33 SEQ ID PATENT201703002, Lachnospiraceae (Genus) (A) NO: 13PATENT201703003, (Clostridium B-67266 Cluster XIVa) (Family + Cluster)Bacillus Ascusbbr_33 SEQ ID PATENT201703001, Lachnospiraceae (Genus) (B)NO: 374 (Clostridium Cluster XIVa) (Family + Cluster) LactobacillusAscusbbr_25200 SEQ ID PATENT201703001, Lachnospiracea (Genus) NO: 14PATENT201703002, incertaesedis PATENT201703003, (Genus) PATENT201703004Lachnospiracea incertaesedis (Genus) Subdoligranulum Ascusbbr_84 SEQ IDPTA-124039, Ruminococcaceae (Genus) NO: 15 PATENT201703003 (ClostridiumCluster III) (Family + Cluster) Subdoligranulum Ascusbbr_136 SEQ IDPTA-124016 Lactobacillus (Genus) NO: 16 (Genus) LachnospiraceaeAscusbbr_128 SEQ ID PATENT201703004 Streptococcus Clostridium NO: 17(Genus) Cluster XIVa (Family + Cluster) Lactobacillus Ascusbbr_322104SEQ ID PATENT201703001 Lachnospiraceae (Genus) NO: 18 (ClostridiumCluster XIVa) (Family + Cluster) Lactobacillus Ascusbbr_409 SEQ IDPTA-124039, Lactobacillus (Genus) (A) NO: 19 PATENT201703002, (Genus)PATENT201703003, Lactobacillus Ascusbbr_409 SEQ ID PATENT201703004(Genus) (B) NO: 375 Lactobacillus Ascusbbr_409 SEQ ID PATENT201703001(Genus) (C) NO: 376 Lactobacillus Ascusbbr_409 SEQ ID PATENT201703001Lactobacillus (Genus) (D) NO: 377 (Genus) Leuconostoc Ascusbbr_127 SEQID B-67265 Paracoccus (Genus) NO: 20 (Genus) LachnospiraceaAscusbbr_14834 SEQ ID PTA-124016, Cellulosilyticum incertae sedis NO: 21PTA-124039, (Genus) (Genus) PATENT201703001, PATENT201703002,PATENT201703003 Lactobacillus Ascusbbr_331885 SEQ ID PTA-124039, Blautia(Genus) (A) NO: 22 PATENT201703002, (Genus) PATENT201703003, B-67269Lactobacillus Ascusbbr_331885 SEQ ID PATENT201703001 (Genus) (B) NO: 378Lactobacillus Ascusbbr_331885 SEQ ID PATENT201703004 (Genus) (C) NO: 379Anaerofilum Ascusbbr_31 SEQ ID PTA-124039, Corynebacterium (Genus) NO:23 PATENT201703002 (Genus) Lachnospiracea Ascusbbr_2307 SEQ IDPTA-124039 Lachnospiracea incertae sedis NO: 24 incertae (Genus) sedis(Genus) Lachnospiraceae Ascusbbr_247 SEQ ID PTA-124039, CorynebacteriumClostridium (A) NO: 25 PATENT201703004 (Genus) Cluster XIVa (Family +Cluster) Lachnospiraceae Ascusbbr_247 SEQ ID PTA-124016 Clostridium (B)NO: 380 Cluster XIVa (Family + Cluster) Microbacterium Ascusbbr_19 SEQID PATENT201703001, Corynebacterium (Genus) NO: 26 B-67264 (Genus)Verrucosispora Ascusbbr_69 SEQ ID Ruminococcus (Genus) NO: 27 (Genus)Anaerofilum Ascusbbr_94 SEQ ID PATENT201703001, Lactobacillus (Genus)NO: 28 PATENT201703004 (Genus) Clostridium Ascusbbr_313454 SEQ IDPATENT201703003, Roseburia sensu stricto NO: 29 PATENT201703004 (Genus)(Genus) Lactobacillus Ascusbbr_351000 SEQ ID Lachnospiraceae (Genus) NO:30 (Clostridium Cluster XIVa) (Family + Cluster) LactobacillusAscusbbr_1436 SEQ ID PTA-124039, Lachnospiraceae (Genus) (A) NO: 31PATENT201703001, (Clostridium PATENT201703002, Cluster PATENT201703003,XIVb) PATENT201703004 (Family + Cluster) Lactobacillus Ascusbbr_1436 SEQID PTA-124016 (Genus) (B) NO: 383 Lachnospiraceae Ascusbbr_28 SEQ IDPTA-124016, Clostridium Clostridium NO: 32 PTA-124039, sensu strictoCluster XIVa PATENT201703002 (Genus) (Family + Cluster) Blautia (Genus)Ascusbbr_144 SEQ ID PTA-124039, Bacteroidesdorei NO: 33 PATENT201703002Lactobacillus Ascusbbr_42760 SEQ ID PTA-124039, Lactobacillus (Genus)(A) NO: 34 PATENT201703002, (Genus) PATENT201703003, PATENT201703004Lactobacillus Ascusbbr_42760 SEQ ID PATENT201703001 (Genus) (B) NO: 384Lactobacillus Ascusbbr_134994 SEQ ID Lactobacillus (Genus) NO: 35(Genus) Lactobacillus Ascusbbr_358773 SEQ ID Nectriaceae (Genus) NO: 36(Family) Pseudomonas Ascusbbr_2503 SEQ ID PATENT201703001 Filobasidium(Genus) NO: 37 floriforme (Genus + species) Sporobacter Ascusbbr_312 SEQID PATENT201703002 Gibberellazeae (Genus) NO: 39 (Genus + species)Lactobacillus Ascusbbr_140914 SEQ ID Alatospora (Genus) NO: 40 (Genus)Lactobacillus Ascusbbr_257627 SEQ ID Hypocreaceae (Genus) NO: 41(Family) Lactobacillus Ascusbbr_310088 SEQ ID Pichia (Genus) NO: 42fermentans (Genus + species) Lachnospiracea Ascusbbr_91 SEQ IDPTA-124016, Candida incertae sedis NO: 43 PTA-124039, railenensis(Genus) PATENT201703002, (Genus + PATENT201703003 species) LactobacillusAscusbbr_150100 SEQ ID Hypocreaceae (Genus) NO: 44 (Family)Lactobacillus Ascusbbr_252028 SEQ ID Lactobacillus (Genus) NO: 45(Genus) Clostridium Ascusbbr_2676 SEQ ID PATENT201703003 Clostridiumbeijerinckii NO: 386 PATENT201703004 saccharolyticum (Genus + (Genus +species) species) Sequence Identifier for Predicted Taxa of AssociatedIsolated Microbes Strain Designation Marker Deposit # LactobacillusAscusbbr_2158 SEQ ID PTA-124039 (Genus) NO: 46 LachnospiraceaeAscusbbr_373 SEQ ID Clostridium NO: 47 Cluster XIVa (Family + Cluster)Lactobacillus Ascusbbr_1802 SEQ ID (Genus) NO: 48 Lactobacillus (Genus)Lactobacillus (Genus) Lactobacillus Ascusbbr_107 SEQ ID PATENT201703002(Genus) NO: 49 Lactobacillus (Genus) Lactobacillus Ascusbbr_1727 SEQ ID(Genus) NO: 50 Lactobacillus Ascusbbr_226 SEQ ID PATENT201703003 (Genus)NO: 338 Faecalibacterium Ascusbbr_17 SEQ ID PATENT201703002, (Genus) NO:339 PATENT201703003, PATENT201703004 Lactobacillus Ascusbbr_14690(A) SEQID PTA-124039, (Genus) NO: 340 PATENT201703001, PATENT201703002,PATENT201703003, PATENT201703004 Ascusbbr_14690(B) SEQ ID PTA-124016,NO: 371 Ascusbbr_14690(C) SEQ ID PATENT201703004 NO: 372 HydrogenoanaeroAscusbbr_18 SEQ ID PATENT201703003 bacterium NO: 341 (Genus)Peptostreptococcaceae Ascusbbr_7363 SEQ ID PTA-124016 Clostridium NO:342 Cluster XI (Family + Cluster) Acrocarpospora Ascusbbr_35 SEQ IDPATENT201703002, (Genus) NO: 343 PATENT201703003, PATENT201703004Lactobacillus Ascusbbr_7779 SEQ ID PATENT201703002, (Genus) NO: 344PATENT201703003, PATENT201703004 Bacillus Ascusbbr_10593(A) SEQ IDPTA-124039, (Genus) NO: 345 PATENT201703001, PATENT201703002,PATENT201703003 Bacillus Ascusbbr_10593(B) SEQ ID PTA-124016, (Genus)NO: 381 Lactobacillus Ascusbbr_32731(A) SEQ ID PTA-124016, (Genus) NO:346 PATENT201703001, PATENT201703002 Ascusbbr_32731(B) SEQ ID PTA-124039NO: 382 Subdoligranulum Ascusbbr_359892 SEQ ID PTA-124039 (Genus) NO:347 Subdoligranulum Ascusbbr_25721 SEQ ID (Genus) NO: 348Lachnospiraceae Ascusbbr_72076 SEQ ID PTA-124039, Clostridium NO: 349PATENT201703001, Cluster XIVa PATENT201703002, (Family +PATENT201703003, Cluster) PATENT201703004 Lactobacillus Ascusbbr_6097SEQ ID PTA-124016, (Genus) NO: 350 PTA-124039, PATENT201703002,PATENT201703003, PATENT201703004 Lactobacillus Ascusbbr_265 (A) SEQ IDPATENT201703001, (Genus) NO: 351 PATENT201703002 LactobacillusPATENT201703004 (Genus) Lactobacillus (Genus) Lactobacillus Ascusbbr_265(B) SEQ ID PATENT201703001, (Genus) NO: 385 PATENT201703002,PATENT201703004 Leuconostoc Ascusbbr_323376 SEQ ID PATENT201703002(Genus) NO: 352 Lachnospiracea Ascusbbr_36257 SEQ ID PTA-124039 incertaesedis NO: 353 (Genus) Lactobacillus Ascusbbr_6957 SEQ ID PTA-124039,(Genus) NO: 354 PATENT201703002, PATENT201703003 Lactobacillus (Genus)Lactobacillus (Genus) Anaerofilum Ascusbbr_38 SEQ ID PATENT201703003(Genus) NO: 355 Lachnospiracea Ascusbbr_13398 SEQ ID PTA-124039,incertae sedis NO: 356 PATENT201703003 (Genus) LachnospiraceaeAscusbbr_57 SEQ ID PATENT201703003 Clostridium NO: 357 Cluster XIVa(Family + Cluster) Lachnospiraceae Clostridium Cluster XIVa (Family +Cluster) Microbacterium Ascusbbr_285160 SEQ ID PTA-124039, (Genus) NO:358 PATENT201703001, PATENT201703002, PATENT201703003, PATENT201703004Verrucosispora Ascusbbr_37385 SEQ ID (Genus) NO: 359 AnaerofilumAscusbbr_118124 SEQ ID PATENT201703001 (Genus) NO: 360 ClostridiumAscusbbr_32592 SEQ ID PATENT201703002 sensu stricto NO: 361 (Genus)Lactobacillus Ascusbbr_110856 SEQ ID PATENT201703004 (Genus) NO: 362Lactobacillus Ascusbbr_185064 SEQ ID PTA-124039, (Genus) NO: 363PATENT201703001, PATENT201703002 Lactobacillus (Genus) LachnospiraceaeAscusbbr_3315 SEQ ID Clostridium NO: 364 Cluster XIVa (Family + Cluster)Blautia (Genus) Ascusbbr_578 SEQ ID PTA-124039 NO: 365 LactobacillusAscusbbr_21169 SEQ ID PATENT201703001 (Genus) NO: 366 Lactobacillus(Genus) Lactobacillus Ascusbbr_48584 SEQ ID PATENT201703004 (Genus) NO:367 Lactobacillus Ascusfbr_15 SEQ ID (Genus) NO: 51 PseudomonasAscusfbr_131 SEQ ID (Genus) NO: 52 Sporobacter Ascusfbr_26 SEQ ID(Genus) NO: 53 Lactobacillus Ascusfbr_2616 SEQ ID (Genus) NO: 54Lactobacillus Ascusfbr_12 SEQ ID (Genus) NO: 55 LactobacillusAscusfbr_53 SEQ ID (Genus) NO: 56 Lachnospiracea Ascusfbr_1379 SEQ IDincertae sedis NO: 57 (Genus) Lactobacillus Ascusfbr_122 SEQ ID (Genus)NO: 58 Lactobacillus Ascusbbr_830 SEQ ID PTA-124039 (Genus) NO: 368Clostridium Ascusbbr_105932 SEQ ID PATENT201703001 beijerinckii NO: 387PATENT201703002 (Genus + species)

TABLE 3 Bacteria of the present disclosure. Sequence Sequence Identifierfor Identifier for Predicted Closest Taxa of Strain Associated PredictedClosest Taxa of Strain Associated Isolated Microbes Designation MarkerIsolated Microbes Designation Marker 1. Clostridium XIVb (Cluster)Ascusbbr_6 SEQ ID Actinomyces (Genus) Ascusbbr_2226 SEQ ID NO: 59 NO:199 2. Gemmiger (Genus) Ascusbbr_113 SEQ ID Succiniclasticum (Genus)Ascusbbr_2227 SEQ ID NO: 60 NO: 200 3. Lactobacillus (Genus)Ascusbbr_116 SEQ ID Beijerinckia (Genus) Ascusbbr_2229 SEQ ID NO: 61 NO:201 4. Clostridium XI (Cluster) Ascusbbr_129 SEQ ID Bosea (Genus)Ascusbbr_2235 SEQ ID NO: 62 NO: 202 5. Jeotgalicoccus (Genus)Ascusbbr_265 SEQ ID Sporobacter (Genus) Ascusbbr_2237 SEQ ID NO: 63 NO:203 6. Lactobacillus (Genus) Ascusbbr_275 SEQ ID Facklamia (Genus)Ascusbbr_2251 SEQ ID NO: 64 NO: 204 7. Lactobacillus (Genus)Ascusbbr_343 SEQ ID Acinetobacter (Genus) Ascusbbr_2266 SEQ ID NO: 65NO: 205 8. Lactobacillus (Genus) Ascusbbr_363 SEQ ID Brevundimonas(Genus) Ascusbbr_2284 SEQ ID NO: 66 NO: 206 9. Jeotgalicoccus (Genus)Ascusbbr_399 SEQ ID Ochrobactrum (Genus) Ascusbbr_2285 SEQ ID NO: 67 NO:207 10. Lactobacillus (Genus) Ascusbbr_444 SEQ ID Alcaligenes (Genus)Ascusbbr_2290 SEQ ID NO: 68 NO: 208 11. Jeotgalicoccus (Genus)Ascusbbr_498 SEQ ID Pseudochrobactrum (Genus) Ascusbbr_2291 SEQ ID NO:69 NO: 209 12. Lactobacillus (Genus) Ascusbbr_542 SEQ ID Jeotgalicoccus(Genus) Ascusbbr_2292 SEQ ID NO: 70 NO: 210 13. Lactobacillus (Genus)Ascusbbr_561 SEQ ID Jeotgalicoccus (Genus) Ascusbbr_2293 SEQ ID NO: 71NO: 211 14. Lactobacillus (Genus) Ascusbbr_570 SEQ ID Acinetobacter(Genus) Ascusbbr_2294 SEQ ID NO: 72 NO: 212 15. Corynebacterium (Genus)Ascusbbr_616 SEQ ID Sphingobacterium (Genus) Ascusbbr_2295 SEQ ID NO: 73NO: 213 16. Microbacterium (Genus) Ascusbbr_620 SEQ ID Lachnospiracea(Genus) Ascusbbr_2301 SEQ ID NO: 74 NO: 214 17. Jeotgalicoccus (Genus)Ascusbbr_690 SEQ ID Azospirillum (Genus) Ascusbbr_2302 SEQ ID NO: 75 NO:215 18. Jeotgalicoccus (Genus) Ascusbbr_705 SEQ ID Lactobacillus (Genus)Ascusbbr_2313 SEQ ID NO: 76 NO: 216 19. Glycomyces (Genus) Ascusbbr_793SEQ ID Clavibacter (Genus) Ascusbbr_2320 SEQ ID NO: 77 NO: 217 20.Streptomyces (Genus) Ascusbbr_795 SEQ ID Clostridium XIVa (Cluster)Ascusbbr_2324 SEQ ID NO: 78 NO: 218 21. Saccharopolyspora (Genus)Ascusbbr_796 SEQ ID Clostridium XIVa (Cluster) Ascusbbr_2325 SEQ ID NO:79 NO: 219 22. Brevibacterium (Genus) Ascusbbr_803 SEQ ID Lactobacillus(Genus) Ascusbbr_2328 SEQ ID NO: 80 NO: 220 23. Microbacterium (Genus)Ascusbbr_804 SEQ ID Clostridium XIVa (Cluster) Ascusbbr_2331 SEQ ID NO:81 NO: 221 24. Acinetobacter (Genus) Ascusbbr_840 SEQ ID Bacillus(Genus) Ascusbbr_2337 SEQ ID NO: 82 NO: 222 25. Lactococcus (Genus)Ascusbbr_846 SEQ ID Methanoplanus (Genus) Ascusbbr_2354 SEQ ID NO: 83NO: 223 26. Cloacibacterium (Genus) Ascusbbr_867 SEQ ID Mogibacterium(Genus) Ascusbbr_2361 SEQ ID NO: 84 NO: 224 27. Mycobacterium (Genus)Ascusbbr_929 SEQ ID Brachybacterium (Genus) Ascusbbr_2368 SEQ ID NO: 85NO: 225 28. Leucobacter (Genus) Ascusbbr_944 SEQ ID Facklamia (Genus)Ascusbbr_2376 SEQ ID NO: 86 NO: 226 29. Lactobacillus (Genus)Ascusbbr_950 SEQ ID Clostridium XIVa (Cluster) Ascusbbr_2378 SEQ ID NO:87 NO: 227 30. Rothia (Genus) Ascusbbr_951 SEQ ID Clostridium XIVa(Cluster) Ascusbbr_2380 SEQ ID NO: 88 NO: 228 31. Lactobacillus (Genus)Ascusbbr_996 SEQ ID Syntrophomonas (Genus) Ascusbbr_2383 SEQ ID NO: 89NO: 229 32. Clavibacter (Genus) Ascusbbr_1005 SEQ ID Beijerinckia(Genus) Ascusbbr_2386 SEQ ID NO: 90 NO: 230 33. Hydrogenoanaerobacterium(Genus) Ascusbbr_1029 SEQ ID Lactobacillus (Genus) Ascusbbr_2390 SEQ IDNO: 91 NO: 231 34. Howardella (Genus) Ascusbbr_1036 SEQ ID Lactobacillus(Genus) Ascusbbr_2391 SEQ ID NO: 92 NO: 232 35. Clostridium (Genus)Ascusbbr_1069 SEQ ID Lactobacillus (Genus) Ascusbbr_2395 SEQ ID NO: 93NO: 233 36. Ascusbbr_1128 SEQ ID Erysipelotrichaceae (Family)Ascusbbr_2397 SEQ ID NO: 94 NO: 234 37. Hydrogenoanaerobacterium (Genus)Ascusbbr_1139 SEQ ID Rummeliibacillus (Genus) Ascusbbr_2399 SEQ ID NO:95 NO: 235 38. Papillibacter (Genus) Ascusbbr_1169 SEQ ID Acinetobacter(Genus) Ascusbbr_2402 SEQ ID NO: 96 NO: 236 39. Butyricicoccus (Genus)Ascusbbr_1185 SEQ ID Lactococcus (Genus) Ascusbbr_2403 SEQ ID NO: 97 NO:237 40. Eubacterium (Genus) Ascusbbr_1245 SEQ ID Propionibacterium(Genus) Ascusbbr_2412 SEQ ID NO: 98 NO: 238 41. Turicibacter (Genus)Ascusbbr_1258 SEQ ID Clostridium (Genus) Ascusbbr_2413 SEQ ID NO: 99 NO:239 42. Lactobacillus (Genus) Ascusbbr_1264 SEQ ID Clostridium XIVa(Cluster) Ascusbbr_2416 SEQ ID NO: 100 NO: 240 43. Asaccharobacter(Genus) Ascusbbr_1332 SEQ ID Rummeliibacillus (Genus) Ascusbbr_2419 SEQID NO: 101 NO: 241 44. Faecalibacterium (Genus) Ascusbbr_1360 SEQ IDRalstonia (Genus) Ascusbbr_2420 SEQ ID NO: 102 NO: 242 45. ClostridiumXIVa (Cluster) Ascusbbr_1363 SEQ ID Brachybacterium (Genus)Ascusbbr_2421 SEQ ID NO: 103 NO: 243 46. Clostridium XIVa (Cluster)Ascusbbr_1422 SEQ ID Ruminobacter (Genus) Ascusbbr_2423 SEQ ID NO: 104NO: 244 47. Clostridium IV (Cluster) Ascusbbr_1424 SEQ ID Glycomyces(Genus) Ascusbbr_2427 SEQ ID NO: 105 NO: 245 48. Clostridium XIVb(Cluster) Ascusbbr_1433 SEQ ID Psychrobacter (Genus) Ascusbbr_2428 SEQID NO: 106 NO: 246 49. Butyricicoccus (Genus) Ascusbbr_1456 SEQ IDYaniella (Genus) Ascusbbr_2429 SEQ ID NO: 107 NO: 247 50. Sporobacter(Genus) Ascusbbr_1485 SEQ ID Clostridium IV (Cluster) Ascusbbr_2431 SEQID NO: 108 NO: 248 51. Butyricicoccus (Genus) Ascusbbr_1488 SEQ IDClostridium (Genus) Ascusbbr_2434 SEQ ID NO: 109 NO: 249 52.Hydrogenoanaerobacterium (Genus) Ascusbbr_1490 SEQ ID Clostridium XIVa(Cluster) Ascusbbr_2435 SEQ ID NO: 110 NO: 250 53. Anaerofilum (Genus)Ascusbbr_1493 SEQ ID Clostridium (Genus) Ascusbbr_2436 SEQ ID NO: 111NO: 251 54. Clostridium XIVa (Cluster) Ascusbbr_1536 SEQ ID ClostridiumXIVa (Cluster) Ascusbbr_2437 SEQ ID NO: 112 NO: 252 55. Clostridium XIVa(Cluster) Ascusbbr_1541 SEQ ID Cohnella (Genus) Ascusbbr_2438 SEQ ID NO:113 NO: 253 56. Lachnospiracea (Family) Ascusbbr_1572 SEQ IDChthonomonas (Genus) Ascusbbr_2441 SEQ ID NO: 114 NO: 254 57.Lachnospiracea (Family) Ascusbbr_1592 SEQ ID Streptophyta (UnrankedAscusbbr_2445 SEQ ID NO: 115 Clade) NO: 255 58. Butyricicoccus (Genus)Ascusbbr_1611 SEQ ID Acinetobacter (Genus) Ascusbbr_2452 SEQ ID NO: 116NO: 256 59. Pediococcus (Genus) Ascusbbr_1614 SEQ ID Clostridium XIVbAscusbbr_2456 SEQ ID NO: 117 NO: 257 60. Acetanaerobacterium (Genus)Ascusbbr_1616 SEQ ID Neisseria Ascusbbr_2465 SEQ ID NO: 118 NO: 258 61.Hydrogenoanaerobacterium (Genus) Ascusbbr_1623 SEQ ID ButyricicoccusAscusbbr_2471 SEQ ID NO: 119 NO: 259 62. Butyricicoccus (Genus)Ascusbbr_1625 SEQ ID Sporobacter Ascusbbr_2472 SEQ ID NO: 120 NO: 26063. Lachnospiracea (Family) Ascusbbr_1632 SEQ ID SporobacterAscusbbr_2476 SEQ ID NO: 121 NO: 261 64. Erysipelotrichaceae (Family)Ascusbbr_1634 SEQ ID Syntrophomonas Ascusbbr_2477 SEQ ID NO: 122 NO: 26265. Lachnospiracea (Family) Ascusbbr_1635 SEQ ID DesulfotomaculumAscusbbr_2478 SEQ ID NO: 123 NO: 263 66. Butyricicoccus (Genus)Ascusbbr_1646 SEQ ID Streptophyta Ascusbbr_2482 SEQ ID NO: 124 NO: 26467. Butyricicoccus (Genus) Ascusbbr_1669 SEQ ID AcetomicrobiumAscusbbr_2489 SEQ ID NO: 125 NO: 265 68. Butyricicoccus (Genus)Ascusbbr_1670 SEQ ID Acinetobacter Ascusbbr_2492 SEQ ID NO: 126 NO: 26669. Butyricicoccus (Genus) Ascusbbr_1674 SEQ ID ErysipelotrichaceaeAscusbbr_2493 SEQ ID NO: 127 NO: 267 70. Butyricicoccus (Genus)Ascusbbr_1678 SEQ ID Jeotgalicoccus Ascusbbr_2496 SEQ ID NO: 128 NO: 26871. Lachnospiracea (Family) Ascusbbr_1679 SEQ ID SelenomonasAscusbbr_2497 SEQ ID NO: 129 NO: 269 72. Howardella (Genu) Ascusbbr_1684SEQ ID Howardella Ascusbbr_2498 SEQ ID NO: 130 NO: 270 73.Lachnospiracea (Family) Ascusbbr_1685 SEQ ID Clostridium XIVaAscusbbr_2500 SEQ ID NO: 131 NO: 271 74. Clavibacter (Genus)Ascusbbr_1694 SEQ ID Lachnospiracea Ascusbbr_2501 SEQ ID NO: 132 NO: 27275. Butyricicoccus (Genus) Ascusbbr_1695 SEQ ID LachnospiraceaAscusbbr_2504 SEQ ID NO: 133 NO: 273 76. Hydrogenoanaerobacterium(Genus) Ascusbbr_1715 SEQ ID Clostridium XIVa Ascusbbr_2506 SEQ ID NO:134 NO: 274 77. Spiroplasma (Genus) Ascusbbr_1720 SEQ ID LachnospiraceaAscusbbr_2508 SEQ ID NO: 135 NO: 275 78. Clostridium XIVa (Cluster)Ascusbbr_1722 SEQ ID Bacillus Ascusbbr_2509 SEQ ID NO: 136 NO: 276 79.Jeotgalicoccus (Genus) Ascusbbr_1723 SEQ ID Paenibacillus Ascusbbr_2510SEQ ID NO: 137 NO: 277 80. Syntrophomonas (Genus) Ascusbbr_1743 SEQ IDEubacterium Ascusbbr_2511 SEQ ID NO: 138 NO: 278 81. Clostridium IV(Cluster) Ascusbbr_1746 SEQ ID Amphibacillus Ascusbbr_2512 SEQ ID NO:139 NO: 279 82. Lachnospiracea (Family) Ascusbbr_1748 SEQ IDStaphylococcus Ascusbbr_2513 SEQ ID NO: 140 NO: 280 83.Hydrogenoanaerobacterium (Genus) Ascusbbr_1753 SEQ ID PaenibacillusAscusbbr_2514 SEQ ID NO: 141 NO: 281 84. Oscillibacter (Genus)Ascusbbr_1756 SEQ ID Clostridium IV Ascusbbr_2515 SEQ ID NO: 142 NO: 28285. Clostridium IV (Cluster) Ascusbbr_1785 SEQ ID PrevotellaAscusbbr_2516 SEQ ID NO: 143 NO: 283 86. Sporobacter (Genus)Ascusbbr_1812 SEQ ID Barnesiella Ascusbbr_2518 SEQ ID NO: 144 NO: 28487. Pediococcus (Genus) Ascusbbr_1821 SEQ ID Clostridium XIVaAscusbbr_2519 SEQ ID NO: 145 NO: 285 88. Sporobacter (Genus)Ascusbbr_1824 SEQ ID Clostridium XIVa Ascusbbr_2520 SEQ ID NO: 146 NO:286 89. Bacillus (Genus) Ascusbbr_1866 SEQ ID Sharpea Ascusbbr_2521 SEQID NO: 147 NO: 287 90. Cellulomonas (Genus) Ascusbbr_1882 SEQ IDLachnospiracea Ascusbbr_2522 SEQ ID NO: 148 NO: 288 91. Syntrophomonas(Genus) Ascusbbr_1887 SEQ ID Leucobacter Ascusbbr_2523 SEQ ID NO: 149NO: 289 92. Cryptanaerobacter (Genus) Ascusbbr_1928 SEQ ID LactonifactorAscusbbr_2524 SEQ ID NO: 150 NO: 290 93. Sporobacter (Genus)Ascusbbr_1932 SEQ ID Lachnospiracea Ascusbbr_2525 SEQ ID NO: 151 NO: 29194. Hydrogenoanaerobacterium (Genus) Ascusbbr_1933 SEQ IDSucciniclasticum Ascusbbr_2526 SEQ ID NO: 152 NO: 292 95. Clostridium IV(Cluster) Ascusbbr_1937 SEQ ID Acidovorax Ascusbbr_2528 SEQ ID NO: 153NO: 293 96. Hydrogenoanaerobacterium (Genus) Ascusbbr_1953 SEQ IDAcinetobacter Ascusbbr_2530 SEQ ID NO: 154 NO: 294 97. Spiroplasma(Genus) Ascusbbr_1955 SEQ ID Comamonas Ascusbbr_2531 SEQ ID NO: 155 NO:295 98. Erysipelotrichaceae (Family) Ascusbbr_1956 SEQ ID PrevotellaAscusbbr_2533 SEQ ID NO: 156 NO: 296 99. Pseudoflavonifractor (Genus)Ascusbbr_1957 SEQ ID Clostridium IV Ascusbbr_2534 SEQ ID NO: 157 NO: 297100. Clostridium XIVa (Cluster) Ascusbbr_1967 SEQ ID ClostridiumAscusbbr_2535 SEQ ID NO: 158 NO: 298 101. Mogibacterium (Genus)Ascusbbr_1969 SEQ ID Succiniclasticum Ascusbbr_2536 SEQ ID NO: 159 NO:299 102. Clostridium (Genus) Ascusbbr_1973 SEQ ID LachnospiraceaAscusbbr_2538 SEQ ID NO: 160 NO: 300 103. Clostridium IV (Cluster)Ascusbbr_2020 SEQ ID Pedobacter Ascusbbr_2539 SEQ ID NO: 161 NO: 301104. Citrobacter (Genus) Ascusbbr_2023 SEQ ID Clostridium XIIAscusbbr_2540 SEQ ID NO: 162 NO: 302 105. Hydrogenoanaerobacterium(Genus) Ascusbbr_2033 SEQ ID Flavobacterium Ascusbbr_2544 SEQ ID NO: 163NO: 303 106. Clostridium XIVa (Cluster) Ascusbbr_2047 SEQ ID ClostridiumAscusbbr_2545 SEQ ID NO: 164 NO: 304 107. Clostridium XIVa (Cluster)Ascusbbr_2049 SEQ ID Alkaliphilus Ascusbbr_2547 SEQ ID NO: 165 NO: 305108. Clostridium (Genus) Ascusbbr_2057 SEQ ID Arthrobacter Ascusbbr_2548SEQ ID NO: 166 NO: 306 109. Erysipelotrichaceae (Family) Ascusbbr_2069SEQ ID Flavobacterium Ascusbbr_2549 SEQ ID NO: 167 NO: 307 110.Clostridium XIVb (Cluster) Ascusbbr_2073 SEQ ID Roseburia Ascusbbr_2550SEQ ID NO: 168 NO: 308 111. Clostridium XIVb (Cluster) Ascusbbr_2076 SEQID Paenibacillus Ascusbbr_2551 SEQ ID NO: 169 NO: 309 112.Butyricicoccus (Genus) Ascusbbr_2101 SEQ ID Olivibacter Ascusbbr_2553SEQ ID NO: 170 NO: 310 113. Pediococcus (Genus) Ascusbbr_2118 SEQ IDClostridium XII Ascusbbr_2554 SEQ ID NO: 171 NO: 311 114. Sphingomonas(Genus) Ascusbbr_2127 SEQ ID Sphingobacterium Ascusbbr_2555 SEQ ID NO:172 NO: 312 115. Clostridium XIVa (Cluster) Ascusbbr_2131 SEQ IDSphingobacterium Ascusbbr_2556 SEQ ID NO: 173 NO: 313 116. ClostridiumIV (Cluster) Ascusbbr_2132 SEQ ID Anaerosporobacter Ascusbbr_2557 SEQ IDNO: 174 NO: 314 117. Clostridium XIVb (Cluster) Ascusbbr_2136 SEQ IDClostridium XII Ascusbbr_2560 SEQ ID NO: 175 NO: 315 118. ClostridiumXIVb (Cluster) Ascusbbr_2137 SEQ ID Clostridium XII Ascusbbr_2561 SEQ IDNO: 176 NO: 316 119. Methylobacterium (Genus) Ascusbbr_2149 SEQ IDClostridium Ascusbbr_2562 SEQ ID NO: 177 NO: 317 120. Salana (Genus)Ascusbbr_2177 SEQ ID Pedobacter Ascusbbr_2563 SEQ ID NO: 178 NO: 318121. Petrobacter (Genus) Ascusbbr_2178 SEQ ID Bacillus Ascusbbr_2564 SEQID NO: 179 NO: 319 122. Bacillus (Genus) Ascusbbr_2180 SEQ IDPaenibacillus Ascusbbr_2565 SEQ ID NO: 180 NO: 320 123. Thermovibrio(Genus) Ascusbbr_2183 SEQ ID Prevotella Ascusbbr_2566 SEQ ID NO: 181 NO:321 124. Erysipelotrichaceae (Family) Ascusbbr_2184 SEQ IDLachnospiracea (Family) Ascusbbr_2567 SEQ ID NO: 182 NO: 322 125.Selenomonas (Genus) Ascusbbr_2192 SEQ ID Lachnospiracea (Family)Ascusbbr_2568 SEQ ID NO: 183 NO: 323 126. Glaciecola (Genus)Ascusbbr_2193 SEQ ID Escherichia/Shigella (Genus) Ascusbbr_2594 SEQ IDNO: 184 NO: 324 127. Lactobacillus (Genus) Ascusbbr_2195 SEQ IDLactobacillus (Genus) Ascusbbr_2603 SEQ ID NO: 185 NO: 325 128.Eubacterium (Genus) Ascusbbr_2200 SEQ ID Corynebacterium (Genus)Ascusbbr_2605 SEQ ID NO: 186 NO: 326 129. Thermomicrobium (Genus)Ascusbbr_2201 SEQ ID Lactobacillus (Genus) Ascusbbr_2615 SEQ ID NO: 187NO: 327 130. Acidobacteria (Genus) Ascusbbr_2204 SEQ ID Lactobacillus(Genus) Ascusbbr_2625 SEQ ID NO: 188 NO: 328 131. Chlorobaculum (Genus)Ascusbbr_2205 SEQ ID Escherichia/Shigella (Genus) Ascusbbr_2640 SEQ IDNO: 189 NO: 329 132. Rothia (Genus) Ascusbbr_2208 SEQ ID Lactobacillus(Genus) Ascusbbr_2644 SEQ ID NO: 190 NO: 330 133. Selenomonas (Genus)Ascusbbr_2210 SEQ ID Lactobacillus (Genus) Ascusbbr_2665 SEQ ID NO: 191NO: 331 134. Clostridium XIVa (Cluster) Ascusbbr_2215 SEQ IDLactobacillus (Genus) Ascusbbr_2684 SEQ ID NO: 192 NO: 332 135.Virgibacillus (Genus) Ascusbbr_2216 SEQ ID Lactobacillus (Genus)Ascusbbr_2694 SEQ ID NO: 193 NO: 333 136. Sphingomonas (Genus)Ascusbbr_2218 SEQ ID Lactobacillus (Genus) Ascusbbr_2699 SEQ ID NO: 194NO: 334 137. Citricoccus (Genus) Ascusbbr_2219 SEQ ID Lactobacillus(Genus) Ascusbbr_2709 SEQ ID NO: 195 NO: 335 138. Catenibacterium(Genus) Ascusbbr_2220 SEQ ID Lactobacillus (Genus) Ascusbbr_2710 SEQ IDNO: 196 NO: 336 139. Amycolatopsis (Genus) Ascusbbr_2224 SEQ IDEnterococcus (Genus) Ascusbbr_2714 SEQ ID NO: 197 NO: 337 140.Sphingobium (Genus) Ascusbbr_2225 SEQ ID Clostridium sensu stricto(Genus) Ascusbbr_2676 SEQ ID NO: 198 NO: 386 141. Clostridium_XIVa(Cluster) Ascusbbr_105932 SEQ ID NO: 387

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a general workflow of one embodiment of the method fordetermining the absolute abundance of one or more active microorganismstrains.

FIG. 2 shows a general workflow of one embodiment of a method fordetermining the co-occurrence of one or more, or two or more, activemicroorganism strains in a sample with one or more metadata(environmental) parameters, followed by leveraging cluster analysis andcommunity detection methods on the network of determined relationships.

FIG. 3 is a graphical representation of the timeline of actions/eventswhich occurred over the 21 study days of the Phase I study whichutilized Cobb 500 broiler chickens.

FIG. 4 is a graphical representation of the timeline of actions/eventswhich occurred over the 21 study days of the Phase II study whichutilized Ross 708 broiler chickens.

FIG. 5 is a graphical representation of an exemplary pen and cage setupfor use in the phase I or II studies described in example I.

FIG. 6 is a graphical representation of an exemplary cage setup for usein the phase I or II studies described in example I.

FIG. 7 is a graphical representation of the summarized datademonstrating the statistical outcomes of treatment 1 vs treatment 2 inthe Phase I study described in Example I.

FIG. 8 is a graphical representation of the summarized datademonstrating the statistical outcomes of treatment 1 vs treatment 2 inthe Phase II study described in Example I.

FIG. 9 is a graphical representation of an exemplary pen setup for usein the study described in Example II, wherein the birds are challengedwith Clostridium perfringens.

FIG. 10 depicts an undegraded carbon source (Day 0) and a degradedcarbon source (Day 7), as utilized in the insoluble carbon sourceassays.

FIG. 11A and FIG. 11B depict the shared percent similarity (percentidentity) among the bacteria (A) and fungi (B) of Table 1. The datapoints represent the greatest percent similarity pairing for eachstrain.

FIG. 12 is a cartoon depiction of an exemplary chicken's anatomy.

FIG. 13 is an image of a dissected gastrointestinal track of a chickenfrom the beak to the cloaca.

FIG. 14 depicts the complex microbial interactions occurring in thegastrointestinal tract. A well-balanced commensal microbial load isinvolved in maintaining multiple homeostatic systems.

FIG. 15 depicts the MIC score distribution for gastrointestinal bacteriaand broiler weight with three species of bacteria and their MIC scores,in which the species have been evaluated in 3^(rd) party studies. Thelower the MIC score, the less likely the species/strains are capable ofpositively modulating broiler weight.

FIG. 16 depicts the MIC score distribution for gastrointestinal bacteriaand broiler feed conversion ration with three species of bacteria andtheir MIC scores, in which the species have been evaluated in 3^(rd)party studies. The lower the MIC score, the less likely thespecies/strains are capable of positively modulating broiler feedconversion ratio.

DETAILED DESCRIPTION

Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

The term “a” or “an” may refer to one or more of that entity, i.e. canrefer to plural referents. As such, the terms “a” or “an”, “one or more”and “at least one” are used interchangeably herein. In addition,reference to “an element” by the indefinite article “a” or “an” does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there is one and only one ofthe elements.

Reference throughout this specification to “one embodiment”, “anembodiment”, “one aspect”, or “an aspect” means that a particularfeature, structure or characteristic described in connection with theembodiment is included in at least one embodiment of the presentdisclosure. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics can be combined inany suitable manner in one or more embodiments.

As used herein, in particular embodiments, the terms “about” or“approximately” when preceding a numerical value indicates the valueplus or minus a range of 10%.

As used herein the terms “microorganism” or “microbe” should be takenbroadly. These terms are used interchangeably and include, but are notlimited to, the two prokaryotic domains, Bacteria and Archaea,eukaryotic fungi and protists, as well as viruses. In some embodiments,the disclosure refers to the “microbes” of Table 1 and/or Table 3, orthe “microbes” incorporated by reference. This characterization canrefer to not only the predicted taxonomic microbial identifiers of thetable, but also the identified strains of the microbes listed in thetable.

The term “microbial ensemble” or “microbial synthetic ensemble” refersto a subset of a microbial community of individual microbial species, orstrains of a species, which can be described as carrying out a commonfunction, or can be described as participating in, or leading to, orcorrelating with, a recognizable parameter, such as a phenotypic traitof interest (e.g. increased feed efficiency in poultry). The communitymay comprise two or more species, or strains of a species, of microbes.In some instances, the microbes coexist within the communitysymbiotically.

The term “microbial community” means a group of microbes comprising twoor more species or strains. Unlike microbial ensembles, a microbialcommunity does not have to be carrying out a common function, or doesnot have to be participating in, or leading to, or correlating with, arecognizable parameter, such as a phenotypic trait of interest (e.g.increased feed efficiency in poultry).

As used herein, “isolate,” “isolated,” “isolated microbe,” and liketerms, are intended to mean that the one or more microorganisms has beenseparated from at least one of the materials with which it is associatedin a particular environment (for example soil, water, animal tissue).

Microbes of the present disclosure may include spores and/or vegetativecells. In some embodiments, microbes of the present disclosure includemicrobes in a viable but non-culturable (VBNC) state, or a quiescentstate. See Liao and Zhao (US Publication US2015267163A1). In someembodiments, microbes of the present disclosure include microbes in abiofilm. See Merritt et al. (U.S. Pat. No. 7,427,408).

Thus, an “isolated microbe” does not exist in its naturally occurringenvironment; rather, it is through the various techniques describedherein that the microbe has been removed from its natural setting andplaced into a non-naturally occurring state of existence. Thus, theisolated strain or isolated microbe may exist as, for example, abiologically pure culture, or as spores (or other forms of the strain)in association with an acceptable carrier.

As used herein, “spore” or “spores” refer to structures produced bybacteria and fungi that are adapted for survival and dispersal. Sporesare generally characterized as dormant structures; however, spores arecapable of differentiation through the process of germination.Germination is the differentiation of spores into vegetative cells thatare capable of metabolic activity, growth, and reproduction. Thegermination of a single spore results in a single fungal or bacterialvegetative cell. Fungal spores are units of asexual reproduction, and insome cases are necessary structures in fungal life cycles. Bacterialspores are structures for surviving conditions that may ordinarily benonconductive to the survival or growth of vegetative cells.

As used herein, “microbial composition” refers to a compositioncomprising one or more microbes of the present disclosure, wherein amicrobial composition, in some embodiments, is administered to animalsof the present disclosure.

As used herein, “carrier”, “acceptable carrier”, or “pharmaceuticalcarrier” refers to a diluent, adjuvant, excipient, or vehicle with whichthe compound is administered. Such carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable, orsynthetic origin; such as peanut oil, soybean oil, mineral oil, sesameoil, and the like. Water or aqueous solution saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, in some embodiments as injectable solutions. Alternatively,the carrier can be a solid dosage form carrier, including but notlimited to one or more of a binder (for compressed pills), a glidant, anencapsulating agent, a flavorant, and a colorant. The choice of carriercan be selected with regard to the intended route of administration andstandard pharmaceutical practice. See Hardee and Baggo (1998.Development and Formulation of Veterinary Dosage Forms. 2^(nd) Ed. CRCPress. 504 pg.); E. W. Martin (1970. Remington's PharmaceuticalSciences. 17^(th) Ed. Mack Pub. Co.); and Blaser et al. (US PublicationUS20110280840A1).

In some aspects, carriers may be granular in structure, such as sand orsand particles. In further aspects, the carriers may be dry, as opposedto a moist or wet carrier. In some aspects, carriers can be nutrititvesubstances and/or prebiotic substances selected fromfructo-oligosaccharides, inulins, isomalto-oligosaccharides, lactitol,lactosucruse, lactulose, pyrodextrines, soy oligosaccharides,transgalacto-oligosaccharides, xylo-oligosaccharides, and vitamins. Insome aspects, carriers can be in solid or liquid form. In some aspects,carriers can be zeolites, calcium carbonate, magnesium carbonate,trehalose, chitosan, shellac, albumin, starch, skim-milk powder,sweet-whey powder, maltodextrin, lactose, and inulin. In some aspects, acarrier is water or physiological saline.

In certain aspects of the disclosure, the isolated microbes exist asisolated and biologically pure cultures. It will be appreciated by oneof skill in the art, that an isolated and biologically pure culture of aparticular microbe, denotes that said culture is substantially free(within scientific reason) of other living organisms and contains onlythe individual microbe in question. The culture can contain varyingconcentrations of said microbe. The present disclosure notes thatisolated and biologically pure microbes often “necessarily differ fromless pure or impure materials.” See, e.g. In re Bergstrom, 427 F.2d1394, (CCPA 1970)(discussing purified prostaglandins), see also, In reBergy, 596 F.2d 952 (CCPA 1979)(discussing purified microbes), see also,Parke-Davis & Co. v. H.K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911)(Learned Hand discussing purified adrenaline), aff'd in part, rev′d inpart, 196 F. 496 (2d Cir. 1912), each of which are incorporated hereinby reference. Furthermore, in some aspects, the disclosure provides forcertain quantitative measures of the concentration, or puritylimitations, that must be found within an isolated and biologically puremicrobial culture. The presence of these purity values, in certainembodiments, is a further attribute that distinguishes the presentlydisclosed microbes from those microbes existing in a natural state. See,e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4thCir. 1958) (discussing purity limitations for vitamin B12 produced bymicrobes), incorporated herein by reference.

As used herein, “individual isolates” should be taken to mean acomposition, or culture, comprising a predominance of a single genera,species, or strain, of microorganism, following separation from one ormore other microorganisms. The phrase should not be taken to indicatethe extent to which the microorganism has been isolated or purified.However, “individual isolates” can comprise substantially only onegenus, species, or strain, of microorganism.

As used herein, “microbiome” refers to the collection of microorganismsthat inhabit the digestive tract or gastrointestinal tract of an animaland the microorganisms' physical environment (i.e., the microbiome has abiotic and physical component). The microbiome is fluid and may bemodulated by numerous naturally occurring and artificial conditions(e.g., change in diet, disease, antimicrobial agents, influx ofadditional microorganisms, etc.). The modulation of the gastrointestinalmicrobiome can be achieved via administration of the compositions of thedisclosure can take the form of: (a) increasing or decreasing aparticular Family, Genus, Species, or functional grouping of a microbe(i.e., alteration of the biotic component of the gastrointestinalmicrobiome) and/or (b) increasing or decreasing gastrointestinal pH,increasing or decreasing volatile fatty acids in the gastrointestinaltract, increasing or decreasing any other physical parameter importantfor gastrointestinal health (i.e., alteration of the abiotic componentof the gut microbiome).

As used herein, “probiotic” refers to a substantially pure microbe(i.e., a single isolate) or a mixture of desired microbes, and may alsoinclude any additional components that can be administered to poultryfor restoring microbiota. Probiotics or microbial inoculant compositionsof the invention may be administered with an agent to allow the microbesto survive the environment of the gastrointestinal tract, i.e., toresist low pH and to grow in the gastrointestinal environment. In someembodiments, the present compositions (e.g., microbial compositions) areprobiotics in some aspects.

As used herein, “prebiotic” refers to an agent that increases the numberand/or activity of one or more desired microbes. Non-limiting examplesof prebiotics that may be useful in the methods of the presentdisclosure include fructooligosaccharides (e.g., oligofructose, inulin,inulin-type fructans), galactooligosaccharides, amino acids, alcohols,isomalto-oligosaccharides, lactitol, lactosucruse, lactulose,pyrodextrines, soy oligosaccharides, transgalacto-oligosaccharides,xylo-oligosaccharides, vitamins, and mixtures thereof. SeeRamirez-Farias et al. (2008. Br. J. Nutr. 4:1-10) and Pool-Zobel andSauer (2007. J. Nutr. 137:2580-2584 and supplemental).

The term “growth medium” as used herein, is any medium which is suitableto support growth of a microbe. By way of example, the media may benatural or artificial including gastrin supplemental agar, LB media,blood serum, and tissue culture gels. It should be appreciated that themedia may be used alone or in combination with one or more other media.It may also be used with or without the addition of exogenous nutrients.

The medium may be amended or enriched with additional compounds orcomponents, for example, a component which may assist in the interactionand/or selection of specific groups of microorganisms. For example,antibiotics (such as penicillin) or sterilants (for example, quaternaryammonium salts and oxidizing agents) could be present and/or thephysical conditions (such as salinity, nutrients (for example organicand inorganic minerals (such as phosphorus, nitrogenous salts, ammonia,potassium and micronutrients such as cobalt and magnesium), pH, and/ortemperature) could be amended.

As used herein, the term “fowl” and “poultry” are used interchangeablyto include both domesticated and non-domesticated birds belonging to theorders of Galliformes and Anseriformes. Fowl include chickens(broilers/fryers/roasters/capons/roosters/stewing hens), turkeys,grouse, New World quail, Old World quail, partridges, ptarmigans,junglefowl, peafowl, ducks, geese, swans, emus, and ostriches.

Broiler chickens of the present disclosure include: Cobb 500, Cobb 700,Cobb Avian 48, Cobb Sasso, Ross 308, Ross 708, Ross PM3, Jersey Giant,Cornish Cross, Delaware, Dorking, Buckeye, Campine, Chantecler,Crevecoeur, Holland, Modern Game, Nankin, Redcap, Russian, Orloff,Spanish, Sultan, Sumatra, Yokohama, Andalusian, Buttercup, Cubalaya,Faverolles, Java, Lakenvelder, Langshan, Malay, Phoenix, Ancona, Aseel,Brahma, Catalana, Cochin, Cornish, Dominique, Hamburg, Houdan, LaFleche, Minorca, New Hampshire, Old English Game, Polish, Rhode IslandWhite, Sebright, Shamo, Australorp, Leghorn, Orpington, Plymouth Rock,Rhode Island Red, Sussex, Wyandotte, Araucana, Iowa Blue, Lamona, ManxRumpy, Naked Neck, Asil, Kadacknath Bursa, Hubbard, Hubbard, Cobb,Hubbard, Lohman, Anak 2000, Avian-34, Starbra, Sam Rat, Bowans, Hyline,BV-300, H & N Nick, Dekalb Lohman, ILI-80, Golden-92, Priya, Sonali,Devendra, B-77, Caribro-91, Varna, Caribro naked necked, Caribromulticolored, Aviagen, Ross, Arbor Acres, Indian River, Peterson,Cobb-Vantress, Avian Sasso, Hybro, Groupe Grimaud, Grimaud Frere,Ameraucana, Silkie, Marans, Rosecomb, Welsummer, Barnevelder, Bantam,Asil, Chantecler, Croad, Houdan, Pekin, Frizzle, Serama, Orloff, Ac,Aseel, Baheij, Bandara, and hybrids thereof.

Egg-laying chickens of the present disclosure include: Ameraucana,Ancona, Andalusian, Appenzeller, Araucana, Australorp, Barnevelder,Brahma, Buckeye, Buttercup, Campine, Catalana, Chantecler, Cochin,Cornish, Crevecoeur, Cubalaya, Deleware, Dominique, Dorking, Faverolles,Fayoumi, Hamburg, Holland, Houdan, Jaerhon, Java, Jersey Giant, LaFleche, Lakenvelder, Lamona, Langsham, Leghorn, Marans, Minorca, NackedNeck, New Hampshire, Orloff, Orpington, Penedesenca, Phoenix, PlymouthRock, Polish, Redcap, Rhode Island, Spanish, Sultan, Sussex, Welsumer,Wyandotte, Yokohama, and hybrids thereof.

While distinctions are made between broiler chickens and egg-layingchickens, embodiments of the present disclosure utilize broilerchickens, egg-laying chickens, and/or multipurpose chickens.

As used herein, “improved” should be taken broadly to encompassimprovement of a characteristic of interest, as compared to a controlgroup, or as compared to a known average quantity associated with thecharacteristic in question. For example, “improved” feed efficiencyassociated with application of a beneficial microbe, or microbialensemble, of the disclosure can be demonstrated by comparing the feedefficiency of poultry treated by the microbes taught herein to the feedefficiency of poultry not treated. In the present disclosure, “improved”does not necessarily demand that the data be statistically significant(i.e. p<0.05); rather, any quantifiable difference demonstrating thatone value (e.g. the average treatment value) is different from another(e.g. the average control value) can rise to the level of “improved.”

As used herein, “inhibiting and suppressing” and like terms should notbe construed to require complete inhibition or suppression, althoughthis may be desired in some embodiments.

The term “marker” or “unique marker” as used herein is an indicator ofunique microorganism type, microorganism strain or activity of amicroorganism strain. A marker can be measured in biological samples andincludes without limitation, a nucleic acid-based marker such as aribosomal RNA gene, a peptide- or protein-based marker, and/or ametabolite or other small molecule marker.

The term “metabolite” as used herein is an intermediate or product ofmetabolism. A metabolite in one embodiment is a small molecule.Metabolites have various functions, including in fuel, structural,signaling, stimulatory and inhibitory effects on enzymes, as a cofactorto an enzyme, in defense, and in interactions with other organisms (suchas pigments, odorants and pheromones). A primary metabolite is directlyinvolved in normal growth, development and reproduction. A secondarymetabolite is not directly involved in these processes but usually hasan important ecological function. Examples of metabolites include butare not limited to antibiotics and pigments such as resins and terpenes,etc. Some antibiotics use primary metabolites as precursors, such asactinomycin which is created from the primary metabolite, tryptophan.Metabolites, as used herein, include small, hydrophilic carbohydrates;large, hydrophobic lipids and complex natural compounds.

As used herein, the term “genotype” refers to the genetic makeup of anindividual cell, cell culture, tissue, organism, or group of organisms.

As used herein, the term “allele(s)” means any of one or morealternative forms of a gene, all of which alleles relate to at least onetrait or characteristic. In a diploid cell, the two alleles of a givengene occupy corresponding loci on a pair of homologous chromosomes.Since the present disclosure, in embodiments, relates to QTLs, i.e.genomic regions that may comprise one or more genes or regulatorysequences, it is in some instances more accurate to refer to “haplotype”(i.e. an allele of a chromosomal segment) instead of “allele”, however,in those instances, the term “allele” should be understood to comprisethe term “haplotype”. Alleles are considered identical when they expressa similar phenotype. Differences in sequence are possible but notimportant as long as they do not influence phenotype.

As used herein, the term “locus” (loci plural) means a specific place orplaces or a site on a chromosome where for example a gene or geneticmarker is found.

As used herein, the term “genetically linked” refers to two or moretraits that are co-inherited at a high rate during breeding such thatthey are difficult to separate through crossing.

A “recombination” or “recombination event” as used herein refers to achromosomal crossing over or independent assortment. The term“recombinant” refers to an organism having a new genetic makeup arisingas a result of a recombination event.

As used herein, the term “molecular marker” or “genetic marker” refersto an indicator that is used in methods for visualizing differences incharacteristics of nucleic acid sequences. Examples of such indicatorsare restriction fragment length polymorphism (RFLP) markers, amplifiedfragment length polymorphism (AFLP) markers, single nucleotidepolymorphisms (SNPs), insertion mutations, microsatellite markers(SSRs), sequence-characterized amplified regions (SCARs), cleavedamplified polymorphic sequence (CAPS) markers or isozyme markers orcombinations of the markers described herein which defines a specificgenetic and chromosomal location. Markers further include polynucleotidesequences encoding 16S or 18S rRNA, and internal transcribed spacer(ITS) sequences, which are sequences found between small-subunit andlarge-subunit rRNA genes that have proven to be especially useful inelucidating relationships or distinctions among when compared againstone another. Mapping of molecular markers in the vicinity of an alleleis a procedure which can be performed by the average person skilled inmolecular-biological techniques.

The primary structure of major rRNA subunit 16S comprise a particularcombination of conserved, variable, and hypervariable regions thatevolve at different rates and enable the resolution of both very ancientlineages such as domains, and more modern lineages such as genera. Thesecondary structure of the 16S subunit include approximately 50 heliceswhich result in base pairing of about 67% of the residues. These highlyconserved secondary structural features are of great functionalimportance and can be used to ensure positional homology in multiplesequence alignments and phylogenetic analysis. Over the previous fewdecades, the 16S rRNA gene has become the most sequenced taxonomicmarker and is the cornerstone for the current systematic classificationof bacteria and archaea (Yarza et al. 2014. Nature Rev. Micro.12:635-45).

A sequence identity of 94.5% or lower for two 16S rRNA genes is strongevidence for distinct genera, 86.5% or lower is strong evidence fordistinct families, 82% or lower is strong evidence for distinct orders,78.5% is strong evidence for distinct classes, and 75% or lower isstrong evidence for distinct phyla. The comparative analysis of 16S rRNAgene sequences enables the establishment of taxonomic thresholds thatare useful not only for the classification of cultured microorganismsbut also for the classification of the many environmental sequences.Yarza et al. 2014. Nature Rev. Micro. 12:635-45).

As used herein, the term “trait” refers to a characteristic orphenotype. For example, in the context of some embodiments of thepresent disclosure; quantity of eggs produced, efficiency of feedutilization, amount of feces produced, susceptibility to gut pathogens,and a decrease in mortality rates, reduced production variability, amongothers. Desirable traits may also include other characteristics,including but not limited to: an increase in weight; an increase in eggproduction; an increase of musculature; an increase of vitamins in eggs;an increase of fatty acid concentration in the gastrointestinal tract;and increase in egg volume; an improved efficiency in feed utilizationand digestibility; an increase in polysaccharide and lignin degradation;an increase in fat, starch, and protein digestion; an increase invitamin availability; an increase in mineral availability; an increasein amino acid availability; improved gastrointestinal development;increasing villi length and surface area; pH balance in thegastrointestinal tract; pH increase in the gastrointestinal tract, pHdecrease in the gastrointestinal tract, a reduction in methane and/ornitrous oxide emissions; a reduction in manure production; an improvedefficiency of nitrogen utilization; an improved efficiency ofphosphorous utilization; an increased resistance to colonization ofpathogenic microbes that colonize chickens; an improvement in meatproperties, reduced mortality, reduced production variability, increasedproduction of antimicrobials, increased clearance of pathogenicmicrobes, increased resistance to colonization of pathogenic microbesthat infect chickens, increased resistance to colonization of pathogenicmicrobes that infect humans improved gut health, etc.; wherein saidincrease, decrease, or reduction is determined by comparing against ananimal not having been administered a composition of the presentdisclosure.

A trait may be inherited in a dominant or recessive manner, or in apartial or incomplete-dominant manner. A trait may be monogenic (i.e.determined by a single locus) or polygenic (i.e. determined by more thanone locus) or may also result from the interaction of one or more geneswith the environment.

In the context of this disclosure, traits may also result from theinteraction of one or more avian genes and one or more microorganismgenes.

As used herein, the term “homozygous” means a genetic condition existingwhen two identical alleles reside at a specific locus, but arepositioned individually on corresponding pairs of homologous chromosomesin the cell of a diploid organism. Conversely, as used herein, the term“heterozygous” means a genetic condition existing when two differentalleles reside at a specific locus, but are positioned individually oncorresponding pairs of homologous chromosomes in the cell of a diploidorganism.

As used herein, the term “phenotype” refers to the observablecharacteristics of an individual cell, cell culture, organism (e.g.,bird), or group of organisms which results from the interaction betweenthat individual's genetic makeup (i.e., genotype) and the environment.

As used herein, the term “chimeric” or “recombinant” when describing anucleic acid sequence or a protein sequence refers to a nucleic acid, ora protein sequence, that links at least two heterologouspolynucleotides, or two heterologous polypeptides, into a singlemacromolecule, or that re-arranges one or more elements of at least onenatural nucleic acid or protein sequence. For example, the term“recombinant” can refer to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques.

As used herein, a “synthetic nucleotide sequence” or “syntheticpolynucleotide sequence” is a nucleotide sequence that is not known tooccur in nature or that is not naturally occurring. Generally, such asynthetic nucleotide sequence will comprise at least one nucleotidedifference when compared to any other naturally occurring nucleotidesequence.

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides, or analogs thereof. This term refers to theprimary structure of the molecule, and thus includes double- andsingle-stranded DNA, as well as double- and single-stranded RNA. It alsoincludes modified nucleic acids such as methylated and/or capped nucleicacids, nucleic acids containing modified bases, backbone modifications,and the like. The terms “nucleic acid” and “nucleotide sequence” areused interchangeably.

As used herein, the term “gene” refers to any segment of DNA associatedwith a biological function. Thus, genes include, but are not limited to,coding sequences and/or the regulatory sequences required for theirexpression. Genes can also include non-expressed DNA segments that, forexample, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

As used herein, the term “homologous” or “homologue” or “ortholog” isknown in the art and refers to related sequences that share a commonancestor or family member and are determined based on the degree ofsequence identity. The terms “homology,” “homologous,” “substantiallysimilar” and “corresponding substantially” are used interchangeablyherein. They refer to nucleic acid fragments wherein changes in one ormore nucleotide bases do not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant disclosure such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the disclosure encompasses more than the specificexemplary sequences. These terms describe the relationship between agene found in one species, subspecies, variety, cultivar or strain andthe corresponding or equivalent gene in another species, subspecies,variety, cultivar or strain. For purposes of this disclosure homologoussequences are compared. “Homologous sequences” or “homologues” or“orthologs” are thought, believed, or known to be functionally related.A functional relationship may be indicated in any one of a number ofways, including, but not limited to: (a) degree of sequence identityand/or (b) the same or similar biological function. Preferably, both (a)and (b) are indicated. Homology can be determined using softwareprograms readily available in the art, such as those discussed inCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30, section 7.718, Table 7.71. Some alignment programsare MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus(Scientific and Educational Software, Pennsylvania) and AlignX (VectorNTI, Invitrogen, Carlsbad, Calif.). Another alignment program isSequencher (Gene Codes, Ann Arbor, Mich.), using default parameters.

As used herein, the term “nucleotide change” refers to, e.g., nucleotidesubstitution, deletion, and/or insertion, as is well understood in theart. For example, mutations contain alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded protein or how the proteins are made.

As used herein, the term “protein modification” refers to, e.g., aminoacid substitution, amino acid modification, deletion, and/or insertion,as is well understood in the art.

As used herein, the term “at least a portion” or “fragment” of a nucleicacid or polypeptide means a portion having the minimal sizecharacteristics of such sequences, or any larger fragment of the fulllength molecule, up to and including the full length molecule. Afragment of a polynucleotide of the disclosure may encode a biologicallyactive portion of a genetic regulatory element. A biologically activeportion of a genetic regulatory element can be prepared by isolating aportion of one of the polynucleotides of the disclosure that comprisesthe genetic regulatory element and assessing activity as describedherein. Similarly, a portion of a polypeptide may be 4 amino acids, 5amino acids, 6 amino acids, 7 amino acids, and so on, going up to thefull length polypeptide. The length of the portion to be used willdepend on the particular application. A portion of a nucleic acid usefulas a hybridization probe may be as short as 12 nucleotides; in someembodiments, it is 20 nucleotides. A portion of a polypeptide useful asan epitope may be as short as 4 amino acids. A portion of a polypeptidethat performs the function of the full-length polypeptide wouldgenerally be longer than 4 amino acids.

Variant polynucleotides also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. Strategiesfor such DNA shuffling are known in the art. See, for example, Stemmer(1994) PNAS 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameriet al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.Biol. 272:336-347; Zhang et al. (1997) PNAS 94:4504-4509; Crameri et al.(1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.For PCR amplifications of the polynucleotides disclosed herein,oligonucleotide primers can be designed for use in PCR reactions toamplify corresponding DNA sequences from cDNA or genomic DNA extractedfrom any organism of interest. Methods for designing PCR primers and PCRcloning are generally known in the art and are disclosed in Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach, thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product is induced, i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH. The (amplification) primer is preferably singlestranded for maximum efficiency in amplification. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact lengths of the primers will depend on manyfactors, including temperature and composition (A/T vs. G/C content) ofprimer. A pair of bi-directional primers consists of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

The terms “stringency” or “stringent hybridization conditions” refer tohybridization conditions that affect the stability of hybrids, e.g.,temperature, salt concentration, pH, formamide concentration and thelike. These conditions are empirically optimized to maximize specificbinding and minimize non-specific binding of primer or probe to itstarget nucleic acid sequence. The terms as used include reference toconditions under which a probe or primer will hybridize to its targetsequence, to a detectably greater degree than other sequences (e.g. atleast 2-fold over background). Stringent conditions are sequencedependent and will be different in different circumstances. Longersequences hybridize specifically at higher temperatures. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of a complementary target sequence hybridizes to aperfectly matched probe or primer. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M Na+ion, typically about 0.01 to 1.0 M Na+ ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes or primers (e.g. 10 to 50 nucleotides) and at least about60° C. for long probes or primers (e.g. greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. Exemplary low stringentconditions or “conditions of reduced stringency” include hybridizationwith a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C. anda wash in 2×SSC at 40° C. Exemplary high stringency conditions includehybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in0.1×SSC at 60° C. Hybridization procedures are well known in the art andare described by e.g. Ausubel et al., 1998 and Sambrook et al., 2001. Insome embodiments, stringent conditions are hybridization in 0.25 MNa2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, 0.5-20% sodium dodecylsulfate at 45° C., such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, followed by awash in 5×SSC, containing 0.1% (w/v) sodium dodecyl sulfate, at 55° C.to 65° C.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Thepromoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity, and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue specificity of apromoter. Promoters may be derived in their entirety from a native gene,or be composed of different elements derived from different promotersfound in nature, or even comprise synthetic DNA segments. It isunderstood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of some variation may have identicalpromoter activity.

As used herein, a “constitutive promoter” is a promoter which is activeunder most conditions and/or during most development stages. There areseveral advantages to using constitutive promoters in expression vectorsused in biotechnology, such as: high level of production of proteinsused to select transgenic cells or organisms; high level of expressionof reporter proteins or scorable markers, allowing easy detection andquantification; high level of production of a transcription factor thatis part of a regulatory transcription system; production of compoundsthat requires ubiquitous activity in the organism; and production ofcompounds that are required during all stages of development.Non-limiting exemplary constitutive promoters include, CaMV 35Spromoter, opine promoters, ubiquitin promoter, alcohol dehydrogenasepromoter, etc.

As used herein, a “non-constitutive promoter” is a promoter which isactive under certain conditions, in certain types of cells, and/orduring certain development stages. For example, tissue specific, tissuepreferred, cell type specific, cell type preferred, inducible promoters,and promoters under development control are non-constitutive promoters.Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues.

As used herein, “inducible” or “repressible” promoter is a promoterwhich is under chemical or environmental factors control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions, certain chemicals, the presenceof light, acidic or basic conditions, etc.

As used herein, a “tissue specific” promoter is a promoter thatinitiates transcription only in certain tissues. Unlike constitutiveexpression of genes, tissue-specific expression is the result of severalinteracting levels of gene regulation. As such, in the art sometimes itis preferable to use promoters from homologous or closely relatedspecies to achieve efficient and reliable expression of transgenes inparticular tissues. This is one of the main reasons for the large amountof tissue-specific promoters isolated from particular tissues found inboth scientific and patent literature.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is regulated by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of regulatingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of thedisclosure can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

As used herein, the phrases “recombinant construct”, “expressionconstruct”, “chimeric construct”, “construct”, and “recombinant DNAconstruct” are used interchangeably herein. A recombinant constructcomprises an artificial combination of nucleic acid fragments, e.g.,regulatory and coding sequences that are not found together in nature.For example, a chimeric construct may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such constructmay be used by itself or may be used in conjunction with a vector. If avector is used then the choice of vector is dependent upon the methodthat will be used to transform host cells as is well known to thoseskilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the disclosure. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others. Vectors can be plasmids, viruses,bacteriophages, pro-viruses, phagemids, transposons, artificialchromosomes, and the like, that replicate autonomously or can integrateinto a chromosome of a host cell. A vector can also be a naked RNApolynucleotide, a naked DNA polynucleotide, a polynucleotide composed ofboth DNA and RNA within the same strand, a poly-lysine-conjugated DNA orRNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or thelike, that is not autonomously replicating. As used herein, the term“expression” refers to the production of a functional end-product e.g.,an mRNA or a protein (precursor or mature).

In some embodiments, the cell or organism has at least one heterologoustrait. As used herein, the term “heterologous trait” refers to aphenotype imparted to a transformed host cell or transgenic organism byan exogenous DNA segment, heterologous polynucleotide or heterologousnucleic acid. Various changes in phenotype are of interest to thepresent disclosure, including but not limited to increasing a fowl'syield of an economically important trait (e.g., eggs, egg volume, fowlweight, etc.) and the like. These results can be achieved by providingexpression of heterologous products or increased expression ofendogenous products in organisms using the methods and compositions ofthe present disclosure. In some embodiments, the isolated microbialstrains of the present disclosure further encompass mutants thereof. Insome embodiments, the present disclosure further contemplates microbialstrains having all of the identifying characteristics of the presentlydisclosed microbial strains.

As used herein, the term “MIC” means maximal information coefficient.MIC is a type of nonparamentric analysis that identifies a score (MICscore) between active microbial strains of the present disclosure and atleast one measured metadata (e.g., increase in weight). Further, U.S.application Ser. No. 15/217,575, filed on Jul. 22, 2016 (issued as U.S.Pat. No. 9,540,676 on Jan. 10, 2017) is hereby incorporated by referencein its entirety.

The maximal information coefficient (MIC) is then calculated betweenstrains and metadata and between strains as seen in FIG. 2, 2009.Results are pooled to create a list of all relationships and theircorresponding MIC scores. If the relationship scores below a giventhreshold, the relationship is deemed/identified as irrelevant. If therelationship is above a given threshold, the relationshipdeemed/identified as relevant, and is further subject to networkanalysis. The following code fragment shows an exemplary methodology forsuch analysis, according to one embodiment:

Read total list of relationships file as links threshold = 0.8 for i inrange(len(links)): if links >= threshold multiplier[i] = 1 elsemultiplier[i] = 0 end if links_temp = multiplier*links final_links =links_temp[links_temp != 0] savetxt(output_file,final_links)output_file.close( )

In some embodiments, the compositions of the present disclosure compriseone or more bacteria and/or one or more fungus that have a MIC score ofat least about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55,0.6, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95.

With regard to MIC scores, and in view of FIG. 15 and FIG. 16. a cut-offbased on this score is used to define useful and non-usefulmicroorganisms with respect to the improvement of specific traits. Thepoint on FIG. 15 and FIG. 16 at which the data points on the curve movetransition from the log scale to the linear scale (with regard to theslope) is the inflection point. The organisms with MIC scores that fallbelow the inflection point are generally non-useful, while the organismswith MIC scores that are found above the inflection point are generallyuseful, as it pertains to the specific characteristic being evaluatedfor the MIC score.

Based on the output of the network analysis, active strains are selectedfor preparing products (e.g., ensembles, aggregates, and/or othersynthetic groupings) containing the selected strains. The output of thenetwork analysis can also be used to inform the selection of strains forfurther product composition testing, as seen in FIG. 2, 2010.

The use of thresholds is discussed above for analyses anddeterminations. Thresholds can be, depending on the implementation andapplication: (1) empirically determined (e.g., based on distributionlevels, setting a cutoff at a number that removes a specified orsignificant portion of low level reads); (2) any non-zero value; (3)percentage/percentile based; (4) only strains whose normalized secondmarker (i.e., activity) reads is greater than normalized first marker(cell count) reads; (5) log 2 fold change between activity and quantityor cell count; (6) normalized second marker (activity) reads is greaterthan mean second marker (activity) reads for entire sample (and/orsample set); and/or any magnitude threshold described above in additionto a statistical threshold (i.e., significance testing). The followingexample provides thresholding detail for distributions of RNA-basedsecond marker measurements with respect to DNA-based first markermeasurements, according to one embodiment.

As used herein “shelf-stable” refers to a functional attribute and newutility acquired by the microbes formulated according to the disclosure,which enable said microbes to exist in a useful/active state outside oftheir natural environment in the gastrointestinal tract (i.e. a markedlydifferent characteristic). Thus, shelf-stable is a functional attributecreated by the formulations/compositions of the disclosure and denotingthat the microbe formulated into a shelf-stable composition can existoutside the gastrointestinal tract and under ambient conditions for aperiod of time that can be determined depending upon the particularformulation utilized, but in general means that the microbes can beformulated to exist in a composition that is stable under ambientconditions for at least a few days and generally at least one week.Accordingly, a “shelf-stable fowl supplement” is a compositioncomprising one or more microbes of the disclosure, said microbesformulated in a composition, such that the composition is stable underambient conditions for at least one week, meaning that the microbescomprised in the composition (e.g. whole cell, spore, or lysed cell) areable to impart one or more beneficial phenotypic properties to a fowlwhen administered (e.g. increased weight gain, increased eggshelldensity, improved gastrointestinal health, and/or modulation of thegastrointestinal microbiome).

Isolated Microbes

In some aspects, the present disclosure provides isolated microbes,including novel strains of microbes, presented in Table 1 and Table 3.

In other aspects, the present disclosure provides isolated wholemicrobial cultures of the microbes identified in Table 1 and Table 3.These cultures may comprise microbes at various concentrations.

In some aspects, the disclosure provides for utilizing one or moremicrobes selected from Table 1 and Table 3 to increase a phenotypictrait of interest in poultry.

In some embodiments, the disclosure provides isolated microbial speciesbelonging to taxonomic families of Lactobacillaceae, Lachnospiraceae,Ruminococcaceae, Peptostreptococcaceae, Streptosporangiaceae,Leuconostocaceae, Microbacteriaceae, Micromonosporaceae, Clostridiaceae,Pseudomonadaceae, Streptococcaceae, Bacillaceae, Bacteroidaceae,Nectriaceae, Corynebacteriaceae, Rhodobacteraceae, and Hypocreaceae.

In further embodiments, isolated microbial species may be selected fromgenera of family Lactobacillaceae, including Acetatifactor,Acetitomaculum, Anaerostipes, Butyrivibrio, Catonella, Cellulosilyticum,Coprococcus, Dorea, Hespellia, Johnsonella, Lachnoanaerobaculum,Lachnobacterium, Lachnospira, Marvinbryantia, Moryella, Oribacterium,Parasporobacterium, Pseudobutyrivibrio, Robinsoniella, Roseburia,Shuttleworthia, Sporobacterium, Stomabaculum, and Syntrophococcus.

In further embodiments, isolated microbial species may be selected fromgenera of family Lachnospiraceae, including Butyrivibrio, Roseburia,Lachnospira, Acetitomaculum, Coprococcus, Johnsonella, Catonella,Pseudobutyrivibrio, Syntrophococcus, Sporobacterium, Parasporobacterium,Lachnobacterium, Shuttleworthia, Dorea, Anaerostipes, Hespellia,Marvinbryantia, Oribacterium, Moryella, Blautia, Robinsoniella,Cellulosilyticum, Lachnoanaerobaculum, Stomatobaculum, Fusicatenibacter,Acetatifactor, and Eisenbergiella.

In further embodiments, isolated microbial species may be selected fromgenera of family Ruminococcaceae, including Ruminococcus, Acetivibrio,Sporobacter, Anaerofilium, Papillibacter, Oscillospira, Gemmiger,Faecalibacterium, Fastidiosipila, Anaerotruncus, Ethanolingenens,Acetanaerobacterium, Subdoligranulum, Hydrogenoanaerobacterium, andCandidadus Soleaferrea.

In further embodiments, isolated microbial species may be selected fromgenera of family Peptostreptococcaceae, including Anaerosphaera,Filifactor, Peptostreptococcus, Sporacetigenium, and Tepidibacter.

In further embodiments, isolated microbial species may be selected fromgenera of family Streptosporangiaceae, including Acrocarpospora,Herbidospora, Microbispora, Microtetraspora, Nonomuraea, Planobispora,Planomonospora, Planotetraspora, Sphaerisporangium, Streptosporangium,Thermoactinospora, Thermocatellispora, and Thermopolyspora.

In further embodiments, isolated microbial species may be selected fromgenera of family Leuconostocaceae, including Fructobacillus,Leuconostoc, Oenococcus, and Weissella.

In further embodiments, isolated microbial species may be selected fromgenera of family Microbacteriaceae, including Agreia, Agrococcus,Agromyces, Alpinomonas, Amnibacterium, Aureobacterium, Chryseoglobus,Clavibacter, Compostimonas, Cryobacterium, Curtobacterium,Diaminobutyricimonas, Frigoribacterium, Frondihabitans, Glacibacter,Gryllotalpicola, Gulosibacter, Herbiconiux, Homoserinimonas, Humibacter,Klugiella, Labedella, Leifsonia, Leucobacter, Lysinimonas,Marisediminicola, Microbacterium, Microcella, Microterricola,Mycetocola, Okibacterium, Phycicola, Plantibacter, Pontimonas,Pseudoclavibacter, Rathayibacter, Rhodoglobus, Salinibacterium,Schumanella, Subtercola, Yonghaparkia, and Zimmermannella.

In further embodiments, isolated microbial species may be selected fromgenera of family Micromonosporaceae, including Actinaurispora,Actinocatenispora, Actinoplanes, Allocatelliglobosispora,Amorphosporangium, Ampullariella, Asanoa, Catelliglobosispora,Catenuloplanes, Couchioplanes, Dactylosporangium, Hamadaea, Jishengella,Krasilnikovia, Longispora, Luedemannella, Micromonospora, Phytohabitans,Phytomonospora, Pilimelia, Planopolyspora, Planosporangium,Plantactinospora, Polymorphospora, Pseudosporangium, Rugosimonospora,Salinispora, Spirilliplanes, Verrucosispora, Virgisporangium, andXiangella.

In further embodiments, isolated microbial species may be selected fromgenera of family Clostridiaceae, including Acetanaerobacterium,Acetivibrio, Acidaminobacter, Alkahphilus, Anaerobacter, Anaerostipes,Anaerotruncus, Anoxynatronum, Bryantella, Butyricicoccus,Caldanaerocella, Caloramator, Caloranaerobacter, Caminicella, CandidatusArthromitus, Clostridium, Coprobacillus, Dorea, Ethanologenbacterium,Faecalibacterium, Garciala, Guggenheimella, Hespellia, Linmingia,Natronincola, Oxobacter, Parasporobacterium, Sarcina, Soehngenia,Sporobacter, Subdoligranulum, Tepidibacter, Tepidimicrobium,Thermobrachium, Thermohalobacter, and Tindallia.

In further embodiments, isolated microbial species may be selected fromgenera of family Pseudomonadaceae.

In further embodiments, isolated microbial species may be selected fromgenera of family Nectriaceae.

In some embodiments, the disclosure provides isolated microbial speciesbelonging to genera of: Hypocreaceae.

In some embodiments, one or more microbes from the taxa disclosed hereinare utilized to impart one or more beneficial properties or improvedtraits to poultry production.

Furthermore, the disclosure relates to microbes having characteristicssubstantially similar to that of a microbe identified in Table 1 and/orTable 3.

The isolated microbial species, and novel strains of said species,identified in the present disclosure, are able to impart beneficialproperties or traits to poultry production.

For instance, the isolated microbes described in Table 1 and Table 3, ormicrobial ensembles of said microbes, are able to increase feedefficiency. The increase can be quantitatively measured, for example, bymeasuring the effect that said microbial application has upon themodulation of feed efficiency. In some embodiments, feed efficiency isrepresented by the feed conversion ratio, which is calculated bymeasuring desirable animal output produced per pound of feed consumed.With regard to fowl, the desirable output is typically pounds of meatproduced per pound of feed consumed.

In some embodiments, the isolated microbial strains are microbes of thepresent disclosure that have been genetically modified. In someembodiments, the genetically modified or recombinant microbes comprisepolynucleotide sequences which do not naturally occur in said microbes.In some embodiments, the microbes may comprise heterologouspolynucleotides. In further embodiments, the heterologouspolynucleotides may be operably linked to one or more polynucleotidesnative to the microbes. In some embodiments, the isolated microbialstrains of the present disclosure further encompass mutants thereof. Insome embodiments, the present disclosure further contemplates microbialstrains having all of the identifying characteristics of the presentlydisclosed microbial strains.

In some embodiments, the heterologous polynucleotides may be reportergenes or selectable markers. In some embodiments, reporter genes may beselected from any of the family of fluorescence proteins (e.g., GFP,RFP, YFP, and the like), β-galactosidase, luciferase. In someembodiments, selectable markers may be selected from neomycinphosphotransferase, hygromycin phosphotransferase, aminoglycosideadenyltransferase, dihydrofolate reductase, acetolactase synthase,bromoxynil nitrilase, β-glucuronidase, dihydrogolate reductase, andchloramphenicol acetyltransferase. In some embodiments, the heterologouspolynucleotide may be operably linked to one or more promoter.

In some embodiments the isolated microbial strains express transgenic ornative enzymes selected from cellulases (endocellulases, exocellulases,glucosidases), pectinases, amylases, amylopectinases, ligninases, andphytases

In some embodiments, the species of the taxa provided in Table 4 are notknown to have been utilized in compositions for administration toanimals.

TABLE 4 Taxa (largely Genera) of the present disclosure not known tohave been utilized in animal agriculture. Corynebacterium VerrucosisporaClostridium XlVa Clostridium Clostridium XI Blautia FaecalibacteriumPseudomonas Hydrogenoanaerobacterium Sporobacter AcrocarposporaClostridium III Subdoligranulum Paracoccus Leuconostoc CellulosilyticumLachnospiracea Ruminococcus Anaerofilum Roseburia MicrobacteriumClostridium XlVb Verrucosispora Bacteroides

Microbial Ensembles

In some aspects, the disclosure provides microbial ensembles comprisinga combination of at least any two microbes selected from amongst themicrobes identified in Table 1 and Table 3.

In certain embodiments, the microbial ensembles of the presentdisclosure comprise two microbes, or three microbes, or four microbes,or five microbes, or six microbes, or seven microbes, or eight microbes,or nine microbes, or ten or more microbes. Said microbes of themicrobial ensembles are different microbial species, or differentstrains of a microbial species.

In some embodiments, the disclosure provides microbial ensembles,comprising: at least one or at least two isolated microbial speciesbelonging to genera of: Lactobacillus, Clostridium, Faecalibacter,Hydrogenoanaerobacterium, Acrocarpospora, Bacillus, Subdoligranulum,Leuconostoc, Lachnospiracea, Anaerofilum, Microbacterium,Verrucosispora, Anaerofilum, Blautia, Pseudomonas, Sporobacter,Corynebacterium, Streptococcus, Paracoccus, Cellulosilyticum,Ruminococcus, Rosebura, Bacteroides, Filobasidium, Gibberella,Alatospora, Pichia, and Candida. Particular novel strains of species ofthese aforementioned genera can be found in Table 1 and Table 3.

In some embodiments, the disclosure provides microbial ensembles,comprising: at least one or at least two isolated microbial speciesbelonging to the family of: Lactobacillaceae, Lachnospiraceae,Ruminococcaceae, Peptostreptococcaceae, Streptosporangiaceae,Leuconostocaceae, Microbacteriaceae, Micromonosporaceae, Clostridiaceae,Pseudomonadales, Nectriaceae, and Hypocreaceae; wherein Lachnospiraceaecan be further specific to Clostridium clusters XIVa and XIVb; andwherein Peptostreptococcaceae can be further specific to Clostridiumcluster XI. Particular novel strains of species of these aforementionedgenera can be found in Table 1 and Table 3.

In particular aspects, the disclosure provides microbial ensembles,comprising species as grouped in Tables 5-11. With respect to Tables5-11, the letters A through I represent a non-limiting selection ofmicrobes of the present disclosure, defined as:

A=Strain designation Ascusbbr_578 identified in Table 1;

B=Strain designation Ascusbbr_1436 identified in Table 1;

C=Strain designation Ascusbbr_33 identified in Table 1;

D=Strain designation Ascusbbr_105932 identified in Table 1;

E=Strain designation Ascusbbr_2676 identified in Table 1;

F=Strain designation Ascusbbr_5796 identified in Table 1;

G=Strain designation Ascusbbr_25721 identified in Table 1;

H=Strain designation Ascusbbr_4729 identified in Table 1; and

I=Strain designation Ascusbbr_7363 identified in Table 1.

TABLE 5 Eight and Nine Strain Microbial Ensembles A, B, C, D, E, F, G, HA, B, C, D, E, F, G, I A, B, C, D, E, F, H, I A, B, C, D, E, G, H, I A,B, C, D, F, G, H, I A, B, C, E, F, G, H, I A, B, D, E, F, G, H, I A, C,D, E, F, G, H, I B, C, D, E, F, G, H, I A, B, C, D, E, F, G, H, I

TABLE 6 Seven Strain Microbial Ensembles A, B, C, D, E, F, G A, B, C, D,E, F, H A, B, C, D, E, F, I A, B, C, D, E, G, H A, B, C, D, E, G, I A,B, C, D, E, H, I A, B, C, D, F, G, H A, B, C, D, F, G, I A, B, C, D, F,H, I A, B, C, D, G, H, I A, B, C, E, F, G, H A, B, C, E, F, G, I A, B,C, E, F, H, I A, B, C, E, G, H, I A, B, C, F, G, H, I A, B, D, E, F, G,H A, B, D, E, F, G, I A, B, D, E, F, H, I A, B, D, E, G, H, I A, B, D,F, G, H, I A, B, E, F, G, H, I A, C, D, E, F, G, H A, C, D, E, F, G, IA, C, D, E, F, H, I A, C, D, E, G, H, I A, C, D, F, G, H, I A, C, E, F,G, H, I A, D, E, F, G, H, I B, C, D, E, F, G, H B, C, D, E, F, G, I B,C, D, E, F, H, I B, C, D, E, G, H, I B, C, D, F, G, H, I B, C, E, F, G,H, I B, D, E, F, G, H, I C, D, E, F, G, H, I

TABLE 7 Six Strain Microbial Ensembles A, B, C, D, E, F A, B, C, D, E, GA, B, C, D, E, H A, B, C, D, E, I A, B, C, D, F, G A, B, C, D, F, H A,B, C, D, F, I A, B, C, D, G, H A, B, C, D, G, I A, B, C, D, H, I A, B,C, E, F, G A, B, C, E, F, H A, B, C, E, F, I A, B, C, E, G, H A, B, C,E, G, I A, B, C, E, H, I A, B, C, F, G, H A, B, C, F, G, I A, B, C, F,H, I A, B, C, G, H, I A, B, D, E, F, G A, B, D, E, F, H A, B, D, E, F, IA, B, D, E, G, H A, B, D, E, G, I A, B, D, E, H, I A, B, D, F, G, H A,B, D, F, G, I D, E, F, G, H, I C, E, F, G, H, I A, B, D, F, H, I A, B,D, G, H, I A, B, E, F, G, H A, B, E, F, G, I A, B, E, F, H, I A, B, E,G, H, I A, B, F, G, H, I A, C, D, E, F, G A, C, D, E, F, H A, C, D, E,F, I A, C, D, E, G, H A, C, D, E, G, I A, C, D, E, H, I A, C, D, F, G, HA, C, D, F, G, I A, C, D, F, H, I A, C, D, G, H, I A, C, E, F, G, H A,C, E, F, G, I A, C, E, F, H, I A, C, E, G, H, I A, C, F, G, H, I A, D,E, F, G, H A, D, E, F, G, I A, D, E, F, H, I A, D, E, G, H, I A, D, F,G, H, I A, E, F, G, H, I B, C, D, E, F, G B, C, D, E, F, H B, C, D, E,F, I B, C, D, E, G, H B, C, D, E, G, I B, C, D, E, H, I B, C, D, F, G, HB, C, D, F, G, I B, C, D, F, H, I B, C, D, G, H, I B, C, E, F, G, H B,C, E, F, G, I B, C, E, F, H, I B, C, E, G, H, I B, C, F, G, H, I B, D,E, F, G, H B, D, E, F, G, I B, D, E, F, H, I B, D, E, G, H, I B, D, F,G, H, I B, E, F, G, H, I C, D, E, F, G, H C, D, E, F, G, I C, D, E, F,H, I C, D, E, G, H, I C, D, F, G, H, I

TABLE 8 Five Strain Microbial Ensembles A, B, C, D, E A, B, C, D, F A,B, C, D, G A, B, C, D, H A, B, C, D, I A, B, C, E, F A, B, C, E, G A, B,C, E, H A, B, C, F, H A, B, C, F, G A, B, C, F, I A, B, C, G, H A, B, C,G, I A, B, C, H, I A, B, D, E, F A, B, D, E, G A, B, D, E, I A, B, D, F,G A, B, D, F, H A, B, D, F, I A, B, D, G, H A, B, D, G, I A, B, D, H, IA, B, E, F, G A, B, E, F, I A, B, E, G, H A, B, E, G, I A, B, E, H, I A,B, F, G, H A, B, F, G, I A, B, F, H, I A, B, G, H, I A, C, D, E, G A, C,D, E, H A, C, D, E, I A, C, D, F, G A, C, D, F, H A, C, D, F, I A, C, D,G, H A, C, D, G, I A, C, E, F, G A, C, E, F, H A, C, E, F, I A, C, E, G,H A, C, E, G, I A, C, E, H, I A, C, F, G, H A, C, F, G, I A, C, G, H, IA, D, E, F, G A, D, E, F, H A, D, E, F, I A, D, E, G, H A, D, E, G, I A,D, E, H, I A, D, F, G, H A, D, F, H, I A, D, G, H, I A, E, F, G, H A, E,F, G, I A, E, F, H, I A, E, G, H, I A, F, G, H, I B, C, D, E, F B, C, D,E, H B, C, D, E, I B, C, D, F, G B, C, D, F, H B, C, D, F, I B, C, D, G,H B, C, D, G, I B, C, D, H, I B, C, E, F, H B, C, E, F, I B, C, E, G, HB, C, E, G, I B, C, E, H, I B, C, F, G, H B, C, F, G, I B, C, F, H, I B,D, E, F, G B, D, E, F, H B, D, E, F, I B, D, E, G, H B, D, E, G, I B, D,E, H, I B, D, F, G, H B, D, F, G, I B, D, G, H, I B, E, F, G, H B, E, F,G, I B, E, F, H, I B, E, G, H, I B, F, G, H, I C, D, E, F, G C, D, E, F,H C, D, E, G, H C, D, E, G, I C, D, E, H, I C, D, F, G, H C, D, F, G, IC, D, F, H, I C, D, G, H, I C, E, F, G, H C, E, F, H, I C, E, G, H, I C,F, G, H, I D, E, F, G, H D, E, F, G, I D, E, F, H, I D, E, G, H, I D, F,G, H, I A, B, C, E, I A, B, D, E, H A, B, E, F, H A, C, D, E, F A, C, D,H, I A, C, F, H, I A, D, F, G, I B, C, D, E, G B, C, E, F, G B, C, G, H,I B, D, F, H, I C, D, E, F, I C, E, F, G, I E, F, G, H, I

TABLE 9 Four Strain Microbial Ensembles A, B, C, D A, B, C, E A, B, C, FA, B, C, G A, B, C, H A, B, C, I A, B, D, E A, B, D, F D, G, H, I A, B,D, G A, B, D, H A, B, D, I A, B, E, F A, B, E, G A, B, E, H A, B, E, IA, B, F, G E, F, G, H A, B, F, H A, D, F, H A, D, F, I A, D, G, H A, D,G, I A, D, H, I A, E, F, G A, E, F, H E, F, G, I A, B, F, I A, B, G, HA, B, G, I A, B, H, I A, C, D, E A, C, D, F A, C, D, G A, C, D, H E, F,H, I A, C, D, I A, C, E, F A, C, E, G A, C, E, H A, C, E, I A, C, F, GA, C, F, H A, C, F, I E, G, H, I A, C, G, H A, C, G, I A, C, H, I A, D,E, F A, D, E, G A, D, E, H A, D, E, I A, D, F, G F, G, H, I A, E, F, IA, E, G, H A, E, G, I A, E, H, I A, F, G, H A, F, G, I A, F, H, I A, G,H, I D, E, F, H B, C, D, E B, C, D, F B, C, D, G B, C, D, H B, C, D, IB, C, E, F B, C, E, G B, C, E, H D, E, F, I B, C, E, I B, C, F, G B, C,F, H B, C, F, I B, C, G, H B, C, G, I B, C, H, I B, D, E, F D, E, G, HB, D, E, G B, D, E, H B, D, E, I B, D, F, G B, D, F, H B, D, F, I B, D,G, H B, D, G, I D, E, G, I B, D, H, I B, E, F, G B, E, F, H B, E, F, IB, E, G, H B, E, G, I B, E, H, I B, F, G, H D, E, H, I B, F, G, I B, F,H, I B, G, H, I C, D, E, F C, D, E, G C, D, E, H C, D, E, I C, D, F, GD, F, G, H C, D, F, H C, D, F, I C, D, G, H C, D, G, I C, D, H, I C, E,F, G C, E, F, H C, E, F, I D, F, G, I C, E, G, H C, E, G, I C, E, H, IC, F, G, H C, F, G, I C, F, H, I C, G, H, I D, E, F, G D, F, H, I

TABLE 10 Three Strain Microbial Ensembles A, B, C A, B, D A, B, E A, B,F A, B, G A, B, H A, B, I A, C, D A, C, E G, H, I E, F, H A, C, F A, C,G A, C, H A, C, I A, D, E A, D, F A, D, G A, D, H A, D, I F, H, I E, F,G A, E, F A, E, G A, E, H A, E, I A, F, G A, F, H A, F, I A, G, H A, G,I F, G, I D, H, I A, H, I B, C, D B, C, E B, C, F B, C, G B, C, H B, C,I B, D, E B, D, F F, G, H D, G, I B, D, G B, D, H B, D, I B, E, F B, E,G B, E, H B, E, I B, F, G B, F, H E, H, I E, F, I B, F, I B, G, H B, G,I B, H, I C, D, E C, D, F C, D, G C, D, H C, D, I E, G, I D, G, H C, E,F C, E, G C, E, H C, E, I C, F, G C, F, H C, F, I C, G, H C, G, I E, G,H D, F, I C, H, I D, E, F D, E, G D, E, H D, E, I D, F, G D, F, H

TABLE 11 Two Strain Microbial Ensembles A, B A, C A, D A, E A, F A, G A,H A, I B, C B, D B, E B, F B, G B, H B, I C, D C, E C, F C, G C, H C, ID, E D, F D, G D, H D, I E, F E, G E, H E, I F, G F, H F, I G, H G, I H,I

In some embodiments, the microbial ensembles may be selected from anymember group from Tables 5-11.

Isolated Microbes—Source Material

The microbes of the present disclosure were obtained, among otherplaces, at various locales in the United States from thegastrointestinal tract of poultry.

Isolated Microbes—Microbial Culture Techniques

The microbes of Table 1 and Table 3 were matched to their nearesttaxonomic groups by utilizing classification tools of the RibosomalDatabase Project (RDP) for 16s rRNA sequences and the User-friendlyNordic ITS Ectomycorrhiza (UNITE) database for ITS rRNA sequences.Examples of matching microbes to their nearest taxa may be found in Lanet al. (2012. PLOS one. 7(3):e32491), Schloss and Westcott (2011. Appl.Environ. Microbiol. 77(10):3219-3226), and Koljalg et al. (2005. NewPhytologist. 166(3):1063-1068).

The isolation, identification, and culturing of the microbes of thepresent disclosure can be effected using standard microbiologicaltechniques. Examples of such techniques may be found in Gerhardt, P.(ed.) Methods for General and Molecular Microbiology. American Societyfor Microbiology, Washington, D.C. (1994) and Lennette, E. H. (ed.)Manual of Clinical Microbiology, Third Edition. American Society forMicrobiology, Washington, D.C. (1980), each of which is incorporated byreference.

Isolation can be effected by streaking the specimen on a solid medium(e.g., nutrient agar plates) to obtain a single colony, which ischaracterized by the phenotypic traits described hereinabove (e.g., Grampositive/negative, capable of forming spores aerobically/anaerobically,cellular morphology, carbon source metabolism, acid/base production,enzyme secretion, metabolic secretions, etc.) and to reduce thelikelihood of working with a culture which has become contaminated.

For example, for microbes of the disclosure, biologically pure isolatescan be obtained through repeated subculture of biological samples, eachsubculture followed by streaking onto solid media to obtain individualcolonies or colony forming units. Methods of preparing, thawing, andgrowing lyophilized bacteria are commonly known, for example, Gherna, R.L. and C. A. Reddy. 2007. Culture Preservation, p 1019-1033. In C. A.Reddy, T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt, andL. R. Snyder, eds. American Society for Microbiology, Washington, D.C.,1033 pages; herein incorporated by reference. Thus freeze dried liquidformulations and cultures stored long term at −70° C. in solutionscontaining glycerol are contemplated for use in providing formulationsof the present disclosure.

The microbes of the disclosure can be propagated in a liquid mediumunder aerobic conditions, or alternatively anaerobic conditions. Mediumfor growing the bacterial strains of the present disclosure includes acarbon source, a nitrogen source, and inorganic salts, as well asspecially required substances such as vitamins, amino acids, nucleicacids and the like. Examples of suitable carbon sources which can beused for growing the microbes include, but are not limited to, starch,peptone, yeast extract, amino acids, sugars such as glucose, arabinose,mannose, glucosamine, maltose, and the like; salts of organic acids suchas acetic acid, fumaric acid, adipic acid, propionic acid, citric acid,gluconic acid, malic acid, pyruvic acid, malonic acid and the like;alcohols such as ethanol and glycerol and the like; oil or fat such assoybean oil, rice bran oil, olive oil, corn oil, sesame oil. The amountof the carbon source added varies according to the kind of carbon sourceand is typically between 1 to 100 gram(s) per liter of medium.Preferably, glucose, starch, and/or peptone is contained in the mediumas a major carbon source, at a concentration of 0.1-5% (W/V). Examplesof suitable nitrogen sources which can be used for growing the bacterialstrains of the present disclosure include, but are not limited to, aminoacids, yeast extract, tryptone, beef extract, peptone, potassiumnitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammoniumphosphate, ammonia or combinations thereof. The amount of nitrogensource varies according to the type of nitrogen source, typicallybetween 0.1 to 30 grams per liter of media. The inorganic salts,potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodiumhydrogen phosphate, magnesium sulfate, magnesium chloride, ferricsulfate, ferrous sulfate, ferric chloride, ferrous chloride, manganoussulfate, manganous chloride, zinc sulfate, zinc chloride, cupricsulfate, calcium chloride, sodium chloride, calcium carbonate, sodiumcarbonate can be used alone or in combination. The amount of inorganicacid varies according to the kind of the inorganic salt, typicallybetween 0.001 to 10 grams per liter of medium. Examples of speciallyrequired substances include, but are not limited to, vitamins, nucleicacids, yeast extract, peptone, meat extract, malt extract, dried yeastand combinations thereof. Cultivation can be effected at a temperature,which allows the growth of the microbial strains, essentially, between20° C. and 46° C. In some aspects, a temperature range is 30° C.-39° C.For optimal growth, in some embodiments, the medium can be adjusted topH 6.0-7.4. It will be appreciated that commercially available media mayalso be used to culture the microbial strains, such as Nutrient Broth orNutrient Agar available from Difco, Detroit, Mich. It will beappreciated that cultivation time may differ depending on the type ofculture medium used and the concentration of sugar as a major carbonsource.

In some aspects, cultivation lasts between 24-96 hours. Microbial cellsthus obtained are isolated using methods, which are well known in theart. Examples include, but are not limited to, membrane filtration andcentrifugal separation. The pH may be adjusted using sodium hydroxideand the like and the culture may be dried using a freeze dryer, untilthe water content becomes equal to 4% or less. Microbial co-cultures maybe obtained by propagating each strain as described hereinabove. In someaspects, microbial multi-strain cultures may be obtained by propagatingtwo or more of the strains described hereinabove. It will be appreciatedthat the microbial strains may be cultured together when compatibleculture conditions can be employed.

Isolated Microbes—Microbial Strains

Microbes can be distinguished into a genus based on polyphasic taxonomy,which incorporates all available phenotypic and genotypic data into aconsensus classification (Vandamme et al. 1996. Polyphasic taxonomy, aconsensus approach to bacterial systematics. Microbiol Rev 1996,60:407-438). One accepted genotypic method for defining species is basedon overall genomic relatedness, such that strains which shareapproximately 70% or more relatedness using DNA-DNA hybridization, with5° C. or less ΔT_(m) (the difference in the melting temperature betweenhomologous and heterologous hybrids), under standard conditions, areconsidered to be members of the same species. Thus, populations thatshare greater than the aforementioned 70% threshold can be considered tobe variants of the same species. Another accepted genotypic method fordefining species is to isolate marker genes of the present disclosure,sequence these genes, and align these sequenced genes from multipleisolates or variants. The microbes are interpreted as belonging to thesame species if one or more of the sequenced genes share at least 97%sequence identity.

The 16S or 18S rRNA sequences or ITS sequences are often used for makingdistinctions between species and strains, in that if one of theaforementioned sequences shares less than a specified % sequenceidentity from a reference sequence, then the two organisms from whichthe sequences were obtained are said to be of different species orstrains.

Thus, one could consider microbes to be of the same species, if theyshare at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identityacross the 16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence.

Further, one could define microbial strains of a species, as those thatshare at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identityacross the 16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence.

Sequence identifiers of the present disclosure consist of SEQ IDNOs:1-387. SEQ ID NOs:1-50 and 59-387 are bacterial polynucleotidesequences encoding 16S rRNA. SEQ ID NOs:51-58 are fungal polynucleotidesequences encoding ITS sequences.

In one embodiment, microbial strains of the present disclosure includethose that comprise polynucleotide sequences that share at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any oneof SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 39, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347,348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361,362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375,376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, and 387. In afurther embodiment, microbial strains of the present disclosure includethose that comprise polynucleotide sequences that share at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any oneof SEQ ID NOs:1-387.

Comparisons may also be made with 23S rRNA sequences against referencesequences.

Unculturable microbes often cannot be assigned to a definite species inthe absence of a phenotype determination, the microbes can be given acandidatus designation within a genus provided their 16S or 18S rRNAsequences or ITS sequences subscribes to the principles of identity withknown species.

One approach is to observe the distribution of a large number of strainsof closely related species in sequence space and to identify clusters ofstrains that are well resolved from other clusters. This approach hasbeen developed by using the concatenated sequences of multiple core(house-keeping) genes to assess clustering patterns, and has been calledmultilocus sequence analysis (MLSA) or multilocus sequence phylogeneticanalysis. MLSA has been used successfully to explore clustering patternsamong large numbers of strains assigned to very closely related speciesby current taxonomic methods, to look at the relationships between smallnumbers of strains within a genus, or within a broader taxonomicgrouping, and to address specific taxonomic questions. More generally,the method can be used to ask whether bacterial species exist—that is,to observe whether large populations of similar strains invariably fallinto well-resolved clusters, or whether in some cases there is a geneticcontinuum in which clear separation into clusters is not observed.

In order to more accurately make a determination of genera, adetermination of phenotypic traits, such as morphological, biochemical,and physiological characteristics are made for comparison with areference genus archetype. The colony morphology can include color,shape, pigmentation, production of slime, etc. Features of the cell aredescribed as to shape, size, Gram reaction, extracellular material,presence of endospores, flagella presence and location, motility, andinclusion bodies. Biochemical and physiological features describe growthof the organism at different ranges of temperature, pH, salinity andatmospheric conditions, growth in presence of different sole carbon andnitrogen sources. One of ordinary skill in the art would be reasonablyapprised as to the phenotypic traits that define the genera of thepresent disclosure.

In one embodiment, the microbes taught herein were identified utilizing16S rRNA gene sequences and ITS sequences. It is known in the art that16S rRNA contains hypervariable regions that can providespecies/strain-specific signature sequences useful for bacterialidentification, and that ITS sequences can also providespecies/strain-specific signature sequences useful for fungalidentification.

Phylogenetic analysis using the rRNA genes and/or ITS sequences are usedto define “substantially similar” species belonging to common genera andalso to define “substantially similar” strains of a given taxonomicspecies. Furthermore, physiological and/or biochemical properties of theisolates can be utilized to highlight both minor and significantdifferences between strains that could lead to advantageous behavior inpoultry.

Compositions of the present disclosure may include combinations offungal spores and bacterial spores, fungal spores and bacterialvegetative cells, fungal vegetative cells and bacterial spores, fungalvegetative cells and bacterial vegetative cells. In some embodiments,compositions of the present disclosure comprise bacteria only in theform of spores. In some embodiments, compositions of the presentdisclosure comprise bacteria only in the form of vegetative cells. Insome embodiments, compositions of the present disclosure comprisebacteria in the absence of fungi. In some embodiments, compositions ofthe present disclosure comprise fungi in the absence of bacteria. Insome embodiments, compositions of the present disclosure comprise VBNCbacteria and/or fungi. In some embodiments, compositions of the presentdisclosure include dormant bacteria and/or fungi.

Bacterial spores may include endospores and akinetes. Fungal spores mayinclude statismospores, ballistospores, autospores, aplanospores,zoospores, mitospores, megaspores, microspores, meiospores,chlamydospores, urediniospores, teliospores, oospores, carpospores,tetraspores, sporangiospores, zygospores, basidiospores, ascospores, andasciospores.

In some embodiments, spores of the composition germinate uponadministration to animals of the present disclosure. In someembodiments, spores of the composition germinate only uponadministration to animals of the present disclosure.

Microbial Compositions

In some embodiments, the microbes of the disclosure are combined intomicrobial compositions.

In some embodiments, the microbial compositions include poultry feed,such as cereals (barley, maize, oats, and the like); starches (tapiocaand the like); oilseed cakes; and vegetable wastes. In some embodiments,the microbial compositions include vitamins, minerals, trace elements,emulsifiers, aromatizing products, binders, colorants, odorants,thickening agents, and the like. In some embodiments, the microbialcompositions include one or more of an ionophore; vaccine; antibiotic;antihelmintic; virucide; nematicide; amino acids such as methionine,glycine, and arginine; fish oil; oregano; and biologically activemolecules such as enzymes.

In some embodiments, the microbial compositions of the presentdisclosure are solid. Where solid compositions are used, it may bedesired to include one or more carrier materials including, but notlimited to: mineral earths such as silicas, talc, kaolin, limestone,chalk, clay, dolomite, diatomaceous earth; calcium sulfate; magnesiumsulfate; magnesium oxide; zeolites, calcium carbonate; magnesiumcarbonate; trehalose; chitosan; shellac; albumins; starch; skim-milkpowder; sweet-whey powder; maltodextrin; lactose; inulin; dextrose;products of vegetable origin such as cereal meals, tree bark meal, woodmeal, and nutshell meal.

In some embodiments, the microbial compositions of the presentdisclosure are liquid. In further embodiments, the liquid comprises asolvent that may include water or an alcohol or a saline or carbohydratesolution, and other animal-safe solvents. In some embodiments, themicrobial compositions of the present disclosure include binders such asanimal-safe polymers, carboxymethylcellulose, starch, polyvinyl alcohol,and the like.

In some embodiments, the microbial compositions of the presentdisclosure comprise thickening agents such as silica, clay, naturalextracts of seeds or seaweed, synthetic derivatives of cellulose, guargum, locust bean gum, alginates, and methylcelluloses. In someembodiments, the microbial compositions comprise anti-settling agentssuch as modified starches, polyvinyl alcohol, xanthan gum, and the like.

In some embodiments, the microbial compositions of the presentdisclosure comprise colorants including organic chromophores classifiedas nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine,anthraquinone, azine, diphenylmethane, indamine, indophenol, methine,oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene.In some embodiments, the microbial compositions of the presentdisclosure comprise trace nutrients such as salts of iron, manganese,boron, copper, cobalt, molybdenum and zinc. In some embodiments, themicrobial compositions comprise dyes, both natural and artificial. Insome embodiments, the dye is green in color.

In some embodiments, the microbial compositions of the presentdisclosure comprise an animal-safe virucide, bacteriocide, ornematicide.

In some embodiments, microbial compositions of the present disclosurecomprise saccharides (e.g., monosaccharides, disaccharides,trisaccharides, polysaccharides, oligosaccharides, and the like),polymeric saccharides, lipids, polymeric lipids, lipopolysaccharides,proteins, polymeric proteins, lipoproteins, nucleic acids, nucleic acidpolymers, silica, inorganic salts and combinations thereof. In a furtherembodiment, microbial compositions comprise polymers of agar, agarose,gelrite, and gellan gum, and the like. In some embodiments, microbialcompositions comprise plastic capsules, emulsions (e.g., water and oil),membranes, and artificial membranes. In some embodiments, emulsions orlinked polymer solutions may comprise microbial compositions of thepresent disclosure. See Harel and Bennett (U.S. Pat. No. 8,460,726B2).In one embodiment, the microbial composition comprises glucose. In oneembodiment, formulations of the microbial composition comprise glucose.

In some embodiments, microbial compositions of the present disclosurecomprise one or more oxygen scavengers, denitrifiers, nitrifiers, heavymetal chelators, and/or dechlorinators; and combinations thereof. In oneembodiment, the one or more oxygen scavengers, denitrifiers, nitrifiers,heavy metal chelators, and/or dechlorinators are not chemically activeonce the microbial compositions are mixed with food and/or water to beadministered to the fowl. In one embodiment, the one or more oxygenscavengers, denitrifiers, nitrifiers, heavy metal chelators, and/ordechlorinators are not chemically active when administered to the fowl.

In some embodiments, microbial compositions of the present disclosureoccur in a solid form (e.g., dispersed lyophilized spores) or a liquidform (microbes interspersed in a storage medium). In some embodiments,microbial compositions of the present disclosure are added in dry formto a liquid to form a suspension immediately prior to administration.

In some embodiments, microbial compositions of the present disclosurecomprise one or more preservatives. The preservatives may be in liquidor gas formulations. The preservatives may be selected from one or moreof monosaccharide, disaccharide, trisaccharide, polysaccharide, aceticacid, ascorbic acid, calcium ascorbate, erythorbic acid, iso-ascorbicacid, erythrobic acid, potassium nitrate, sodium ascorbate, sodiumerythorbate, sodium iso-ascorbate, sodium nitrate, sodium nitrite,nitrogen, benzoic acid, calcium sorbate, ethyl lauroyl arginate,methyl-p-hydroxy benzoate, methyl paraben, potassium acetate, potassiumbenzoiate, potassium bisulphite, potassium diacetate, potassium lactate,potassium metabisulphite, potassium sorbate, propyl-p-hydroxy benzoate,propyl paraben, sodium acetate, sodium benzoate, sodium bisulphite,sodium nitrite, sodium diacetate, sodium lactate, sodium metabisulphite,sodium salt of methyl-p-hydroxy benzoic acid, sodium salt ofpropyl-p-hydroxy benzoic acid, sodium sulphate, sodium sulfite, sodiumdithionite, sulphurous acid, calcium propionate, dimethyl dicarbonate,natamycin, potassium sorbate, potassium bisulfite, potassiummetabisulfite, propionic acid, sodium diacetate, sodium propionate,sodium sorbate, sorbic acid, ascorbic acid, ascorbyl palmitate, ascorbylstearate, butylated hydro-xyanisole, butylated hydroxytoluene (BHT),butylated hydroxyl anisole (BHA), citric acid, citric acid esters ofmono- and/or diglycerides, L-cysteine, L-cysteine hydrochloride, gumguaiacum, gum guaiac, lecithin, lecithin citrate, monoglyceride citrate,monoisopropyl citrate, propyl gallate, sodium metabisulphite, tartaricacid, tertiary butyl hydroquinone, stannous chloride, thiodipropionicacid, dilauryl thiodipropionate, distearyl thiodipropionate, ethoxyquin,sulfur dioxide, formic acid, or tocopherol(s).

In some embodiments, microbial compositions of the present disclosureinclude bacterial and/or fungal cells in spore form, vegetative cellform, dormant cell form, and/or lysed form. In one embodiment, the lysedcell form acts as a mycotoxin binder, e.g. mycotoxins binding to deadcells.

In some embodiments, the microbial compositions are shelf stable in arefrigerator (35-40° F.) for a period of at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60days. In some embodiments, the microbial compositions are shelf stablein a refrigerator (35-40° F.) for a period of at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60weeks.

In some embodiments, the microbial compositions are shelf stable at roomtemperature (68-72° F.) or between 50-77° F. for a period of at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, or 60 days. In some embodiments, the microbial compositions areshelf stable at room temperature (68-72° F.) or between 50-77° F. for aperiod of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions are shelf stable at−23-35° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In someembodiments, the microbial compositions are shelf stable at −23-35° F.for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions are shelf stable at77-100° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In someembodiments, the microbial compositions are shelf stable at 77-100° F.for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions are shelf stable at101-213° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In someembodiments, the microbial compositions are shelf stable at 101-213° F.for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of about 1 to100, about 1 to 95, about 1 to 90, about 1 to 85, about 1 to 80, about 1to 75, about 1 to 70, about 1 to 65, about 1 to 60, about 1 to 55, about1 to 50, about 1 to 45, about 1 to 40, about 1 to 35, about 1 to 30,about 1 to 25, about 1 to 20, about 1 to 15, about 1 to 10, about 1 to5, about 5 to 100, about 5 to 95, about 5 to 90, about 5 to 85, about 5to 80, about 5 to 75, about 5 to 70, about 5 to 65, about 5 to 60, about5 to 55, about 5 to 50, about 5 to 45, about 5 to 40, about 5 to 35,about 5 to 30, about 5 to 25, about 5 to 20, about 5 to 15, about 5 to10, about 10 to 100, about 10 to 95, about 10 to 90, about 10 to 85,about 10 to 80, about 10 to 75, about 10 to 70, about 10 to 65, about 10to 60, about 10 to 55, about 10 to 50, about 10 to 45, about 10 to 40,about 10 to 35, about 10 to 30, about 10 to 25, about 10 to 20, about 10to 15, about 15 to 100, about 15 to 95, about 15 to 90, about 15 to 85,about 15 to 80, about 15 to 75, about 15 to 70, about 15 to 65, about 15to 60, about 15 to 55, about 15 to 50, about 15 to 45, about 15 to 40,about 15 to 35, about 15 to 30, about 15 to 25, about 15 to 20, about 20to 100, about 20 to 95, about 20 to 90, about 20 to 85, about 20 to 80,about 20 to 75, about 20 to 70, about 20 to 65, about 20 to 60, about 20to 55, about 20 to 50, about 20 to 45, about 20 to 40, about 20 to 35,about 20 to 30, about 20 to 25, about 25 to 100, about 25 to 95, about25 to 90, about 25 to 85, about 25 to 80, about 25 to 75, about 25 to70, about 25 to 65, about 25 to 60, about 25 to 55, about 25 to 50,about 25 to 45, about 25 to 40, about 25 to 35, about 25 to 30, about 30to 100, about 30 to 95, about 30 to 90, about 30 to 85, about 30 to 80,about 30 to 75, about 30 to 70, about 30 to 65, about 30 to 60, about 30to 55, about 30 to 50, about 30 to 45, about 30 to 40, about 30 to 35,about 35 to 100, about 35 to 95, about 35 to 90, about 35 to 85, about35 to 80, about 35 to 75, about 35 to 70, about 35 to 65, about 35 to60, about 35 to 55, about 35 to 50, about 35 to 45, about 35 to 40,about 40 to 100, about 40 to 95, about 40 to 90, about 40 to 85, about40 to 80, about 40 to 75, about 40 to 70, about 40 to 65, about 40 to60, about 40 to 55, about 40 to 50, about 40 to 45, about 45 to 100,about 45 to 95, about 45 to 90, about 45 to 85, about 45 to 80, about 45to 75, about 45 to 70, about 45 to 65, about 45 to 60, about 45 to 55,about 45 to 50, about 50 to 100, about 50 to 95, about 50 to 90, about50 to 85, about 50 to 80, about 50 to 75, about 50 to 70, about 50 to65, about 50 to 60, about 50 to 55, about 55 to 100, about 55 to 95,about 55 to 90, about 55 to 85, about 55 to 80, about 55 to 75, about 55to 70, about 55 to 65, about 55 to 60, about 60 to 100, about 60 to 95,about 60 to 90, about 60 to 85, about 60 to 80, about 60 to 75, about 60to 70, about 60 to 65, about 65 to 100, about 65 to 95, about 65 to 90,about 65 to 85, about 65 to 80, about 65 to 75, about 65 to 70, about 70to 100, about 70 to 95, about 70 to 90, about 70 to 85, about 70 to 80,about 70 to 75, about 75 to 100, about 75 to 95, about 75 to 90, about75 to 85, about 75 to 80, about 80 to 100, about 80 to 95, about 80 to90, about 80 to 85, about 85 to 100, about 85 to 95, about 85 to 90,about 90 to 100, about 90 to 95, or 95 to 100 weeks.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of 1 to 100, 1to 95, 1 to 90, 1 to 85, 1 to 80, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1to 55, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 5 to 100, 5 to 95, 5 to 90, 5 to 85, 5 to 80, 5to 75, 5 to 70, 5 to 65, 5 to 60, 5 to 55, 5 to 50, 5 to 45, 5 to 40, 5to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 100, 10 to 95,10 to 90, 10 to 85, 10 to 80, 10 to 75, 10 to 70, 10 to 65, 10 to 60, 10to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to20, 10 to 15, 15 to 100, 15 to 95, 15 to 90, 15 to 85, 15 to 80, 15 to75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 100, 20 to 95, 20 to90, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60, 20 to55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to100, 25 to 95, 25 to 90, 25 to 85, 25 to 80, 25 to 75, 25 to 70, 25 to65, 25 to 60, 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to30, 30 to 100, 30 to 95, 30 to 90, 30 to 85, 30 to 80, 30 to 75, 30 to70, 30 to 65, 30 to 60, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to35, 35 to 100, 35 to 95, 35 to 90, 35 to 85, 35 to 80, 35 to 75, 35 to70, 35 to 65, 35 to 60, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to100, 40 to 95, 40 to 90, 40 to 85, 40 to 80, 40 to 75, 40 to 70, 40 to65, 40 to 60, 40 to 55, 40 to 50, 40 to 45, 45 to 100, 45 to 95, 45 to90, 45 to 85, 45 to 80, 45 to 75, 45 to 70, 45 to 65, 45 to 60, 45 to55, 45 to 50, 50 to 100, 50 to 95, 50 to 90, 50 to 85, 50 to 80, 50 to75, 50 to 70, 50 to 65, 50 to 60, 50 to 55, 55 to 100, 55 to 95, 55 to90, 55 to 85, 55 to 80, 55 to 75, 55 to 70, 55 to 65, 55 to 60, 60 to100, 60 to 95, 60 to 90, 60 to 85, 60 to 80, 60 to 75, 60 to 70, 60 to65, 65 to 100, 65 to 95, 65 to 90, 65 to 85, 65 to 80, 65 to 75, 65 to70, 70 to 100, 70 to 95, 70 to 90, 70 to 85, 70 to 80, 70 to 75, 75 to100, 75 to 95, 75 to 90, 75 to 85, 75 to 80, 80 to 100, 80 to 95, 80 to90, 80 to 85, 85 to 100, 85 to 95, 85 to 90, 90 to 100, 90 to 95, or 95to 100 weeks.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of about 1 to36, about 1 to 34, about 1 to 32, about 1 to 30, about 1 to 28, about 1to 26, about 1 to 24, about 1 to 22, about 1 to 20, about 1 to 18, about1 to 16, about 1 to 14, about 1 to 12, about 1 to 10, about 1 to 8,about 1 to 6, about 1 one 4, about 1 to 2, about 4 to 36, about 4 to 34,about 4 to 32, about 4 to 30, about 4 to 28, about 4 to 26, about 4 to24, about 4 to 22, about 4 to 20, about 4 to 18, about 4 to 16, about 4to 14, about 4 to 12, about 4 to 10, about 4 to 8, about 4 to 6, about 6to 36, about 6 to 34, about 6 to 32, about 6 to 30, about 6 to 28, about6 to 26, about 6 to 24, about 6 to 22, about 6 to 20, about 6 to 18,about 6 to 16, about 6 to 14, about 6 to 12, about 6 to 10, about 6 to8, about 8 to 36, about 8 to 34, about 8 to 32, about 8 to 30, about 8to 28, about 8 to 26, about 8 to 24, about 8 to 22, about 8 to 20, about8 to 18, about 8 to 16, about 8 to 14, about 8 to 12, about 8 to 10,about 10 to 36, about 10 to 34, about 10 to 32, about 10 to 30, about 10to 28, about 10 to 26, about 10 to 24, about 10 to 22, about 10 to 20,about 10 to 18, about 10 to 16, about 10 to 14, about 10 to 12, about 12to 36, about 12 to 34, about 12 to 32, about 12 to 30, about 12 to 28,about 12 to 26, about 12 to 24, about 12 to 22, about 12 to 20, about 12to 18, about 12 to 16, about 12 to 14, about 14 to 36, about 14 to 34,about 14 to 32, about 14 to 30, about 14 to 28, about 14 to 26, about 14to 24, about 14 to 22, about 14 to 20, about 14 to 18, about 14 to 16,about 16 to 36, about 16 to 34, about 16 to 32, about 16 to 30, about 16to 28, about 16 to 26, about 16 to 24, about 16 to 22, about 16 to 20,about 16 to 18, about 18 to 36, about 18 to 34, about 18 to 32, about 18to 30, about 18 to 28, about 18 to 26, about 18 to 24, about 18 to 22,about 18 to 20, about 20 to 36, about 20 to 34, about 20 to 32, about 20to 30, about 20 to 28, about 20 to 26, about 20 to 24, about 20 to 22,about 22 to 36, about 22 to 34, about 22 to 32, about 22 to 30, about 22to 28, about 22 to 26, about 22 to 24, about 24 to 36, about 24 to 34,about 24 to 32, about 24 to 30, about 24 to 28, about 24 to 26, about 26to 36, about 26 to 34, about 26 to 32, about 26 to 30, about 26 to 28,about 28 to 36, about 28 to 34, about 28 to 32, about 28 to 30, about 30to 36, about 30 to 34, about 30 to 32, about 32 to 36, about 32 to 34,or about 34 to 36 months.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of 1 to 36 1 to34 1 to 32 1 to 30 1 to 28 1 to 26 1 to 24 1 to 22 1 to 20 1 to 18 1 to16 1 to 14 1 to 12 1 to 10 1 to 8 1 to 6 1 one 4 1 to 24 to 36 4 to 34 4to 32 4 to 30 4 to 28 4 to 26 4 to 24 4 to 22 4 to 20 4 to 18 4 to 16 4to 14 4 to 12 4 to 10 4 to 8 4 to 6 6 to 36 6 to 34 6 to 32 6 to 30 6 to28 6 to 26 6 to 24 6 to 22 6 to 20 6 to 18 6 to 16 6 to 14 6 to 12 6 to10 6 to 8 8 to 36 8 to 34 8 to 32 8 to 30 8 to 28 8 to 26 8 to 24 8 to22 8 to 20 8 to 18 8 to 16 8 to 14 8 to 12 8 to 10 10 to 36 10 to 34 10to 32 10 to 30 10 to 28 10 to 26 10 to 24 10 to 22 10 to 20 10 to 18 10to 16 10 to 14 10 to 12 12 to 36 12 to 34 12 to 32 12 to 30 12 to 28 12to 26 12 to 24 12 to 22 12 to 20 12 to 18 12 to 16 12 to 14 14 to 36 14to 34 14 to 32 14 to 30 14 to 28 14 to 26 14 to 24 14 to 22 14 to 20 14to 18 14 to 16 16 to 36 16 to 34 16 to 32 16 to 30 16 to 28 16 to 26 16to 24 16 to 22 16 to 20 16 to 18 18 to 36 18 to 34 18 to 32 18 to 30 18to 28 18 to 26 18 to 24 18 to 22 18 to 20 20 to 36 20 to 34 20 to 32 20to 30 20 to 28 20 to 26 20 to 24 20 to 22 22 to 36 22 to 34 22 to 32 22to 30 22 to 28 22 to 26 22 to 24 24 to 36 24 to 34 24 to 32 24 to 30 24to 28 24 to 26 26 to 36 26 to 34 26 to 32 26 to 30 26 to 28 28 to 36 28to 34 28 to 32 28 to 30 30 to 36 30 to 34 30 to 32 32 to 36 32 to 34, orabout 34 to 36 months.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at any of the disclosed temperatures and/ortemperature ranges and spans of time at a relative humidity of at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, or 98%.

Moisture content is a measurement of the total amount of water in acomposition, usually expressed as a percentage of the total weight. Themoisture content is a useful measurement for determining the dry weightof a composition, and it can be used to confirm whether thedesiccation/drying process of a composition is complete. The moisturecontent is calculated by dividing the (wet weight of the compositionminus the weight after desiccating/drying) by the wet weight of thecomposition, and multiplying by 100.

Moisture content defines the amount of water in a composition, but wateractivity explains how the water in the composition will react withmicroorganisms. The greater the water activity, the fastermicroorganisms are able to grow. Water activity is calculated by findingthe ratio of the vapor pressure in a composition to the vapor pressureof pure water. More specifically, the water activity is the partialvapor pressure of water in a composition divided by the standard statepartial vapor pressure of pure water. Pure distilled water has a wateractivity of 1. A determination of water activity of a composition is notthe amount of water in a composition, rather it is the amount of excessamount of water that is available for microorganisms to use.Microorganisms have a minimal and optimal water activity for growth.

In some embodiments, the microbial compositions of the presentdisclosure are desiccated. A microbial composition is desiccated if themoisture content of the composition is between 0% and 20%.

In some embodiments, the microbial compositions of the presentdisclosure have a moisture content of about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%,about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%,about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%,about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%,about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%,about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%,about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or about 100%.

In some embodiments, the microbial compositions of the presentdisclosure have a moisture content of less than 0.5%, less than 0.6%,less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than2%, less than 3%, less than 4%, less than 5%, less than 6%, less than7%, less than 8%, less than 9%, less than 10%, less than 11%, less than12%, less than 13%, less than 14%, less than 15%, less than 16%, lessthan 17%, less than 18%, less than 19%, less than 20%, less than 21%,less than 22%, less than 23%, less than 24%, less than 25%, less than26%, less than 27%, less than 28%, less than 29%, less than 30%, lessthan 31%, less than 32%, less than 33%, less than 34%, less than 35%,less than 36%, less than 37%, less than 38%, less than 39%, less than40%, less than 41%, less than 42%, less than 43%, less than 44%, lessthan 45%, less than 46%, less than 4′7%, less than 48%, less than 49%,less than 50%, less than 51%, less than 52%, less than 53%, less than54%, less than 55%, less than 56%, less than 57%, less than 58%, lessthan 59%, less than 60%, less than 61%, less than 62%, less than 63%,less than 64%, less than 65%, less than 66%, less than 67%, less than68%, less than 69%, less than 70%, less than 71%, less than 72%, lessthan 73%, less than 74%, less than 75%, less than 76%, less than 77%,less than 78%, less than 79%, less than 80%, less than 81%, less than82%, less than 83%, less than 84%, less than 85%, less than 86%, lessthan 87%, less than 88%, less than 89%, less than 90%, less than 91%,less than 92%, less than 93%, less than 94%, less than 95%, less than96%, less than 97%, less than 98%, less than 99%, or less than 100%.

In some embodiments, the microbial compositions of the presentdisclosure have a moisture content of less than about 0.5%, less thanabout 0.6%, less than about 0.7%, less than about 0.8%, less than about0.9%, less than about 1%, less than about 2%, less than about 3%, lessthan about 4%, less than about 5%, less than about 6%, less than about7%, less than about 8%, less than about 9%, less than about 10%, lessthan about 11%, less than about 12%, less than about 13%, less thanabout 14%, less than about 15%, less than about 16%, less than about17%, less than about 18%, less than about 19%, less than about 20%, lessthan about 21%, less than about 22%, less than about 23%, less thanabout 24%, less than about 25%, less than about 26%, less than about27%, less than about 28%, less than about 29%, less than about 30% lessthan about 31%, less than about 32%, less than about 33%, less thanabout 34%, less than about 35%, less than about 36%, less than about3′7%, less than about 38%, less than about 39% less than about 40%, lessthan about 41%, less than about 42%, less than about 43%, less thanabout 44%, less than about 45%, less than about 46%, less than about47%, less than about 48% less than about 49%, less than about 50%, lessthan about 51%, less than about 52%, less than about 53%, less thanabout 54%, less than about 55%, less than about 56%, less than about 57%less than about 58%, less than about 59%, less than about 60%, less thanabout 61%, less than about 62%, less than about 63%, less than about64%, less than about 65%, less than about 66% less than about 67%, lessthan about 68%, less than about 69%, less than about 70%, less thanabout 71%, less than about 72%, less than about 73%, less than about74%, less than about 75% less than about 76%, less than about 77%, lessthan about 78%, less than about 79%, less than about 80%, less thanabout 81%, less than about 82%, less than about 83%, less than about84%, less than about 85%, less than about 86%, less than about 87%, lessthan about 88%, less than about 89%, less than about 90%, less thanabout 91%, less than about 92%, less than about 93%, less than about94%, less than about 95%, less than about 96%, less than about 97%, lessthan about 98%, less than about 99%, or less than about 100%.

In some embodiments, the microbial compositions of the presentdisclosure have a moisture content of 1% to 100%, 1% to 95%, 1% to 90%,1% to 85%, 1% to 80%, 1% to 75%, 1% to 70%, 1% to 65%, 1% to 60%, 1% to55%, 1% to 50%, 1% to 45%, 1% to 40%, 1% to 35%, 1% to 30%, 1% to 25%,1% to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 5% to 100%, 5% to 95%, 5% to90%, 5% to 85%, 5% to 80%, 5% to 75%, 5% to 70%, 5% to 65%, 5% to 60%,5% to 55%, 5% to 50%, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to25%, 5% to 20%, 5% to 15%, 5% to 10%, 10% to 100%, 10% to 95%, 10% to90%, 10% to 85%, 10% to 80%, 10% to 75%, 10% to 70%, 10% to 65%, 10% to60%, 10% to 55%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to30%, 10% to 25%, 10% to 20%, 10% to 15%, 15% to 100%, 15% to 95%, 15% to90%, 15% to 85%, 15% to 80%, 15% to 75%, 15% to 70%, 15% to 65%, 15% to60%, 15% to 55%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to 35%, 15% to30%, 15% to 25%, 15% to 20%, 20% to 100%, 20% to 95%, 20% to 90%, 20% to85%, 20% to 80%, 20% to 75%, 20% to 70%, 20% to 65%, 20% to 60%, 20% to55%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, 20% to25%, 25% to 100%, 25% to 95%, 25% to 90%, 25% to 85%, 25% to 80%, 25% to75%, 25% to 70%, 25% to 65%, 25% to 60%, 25% to 55%, 25% to 50%, 25% to45%, 25% to 40%, 25% to 35%, 25% to 30%, 30% to 100%, 30% to 95%, 30% to90%, 30% to 85%, 30% to 80%, 30% to 75%, 30% to 70%, 30% to 65%, 30% to60%, 30% to 55%, 30% to 50%, 30% to 45%, 30% to 40%, 30% to 35%, 35% to100%, 35% to 95%, 35% to 90%, 35% to 85%, 35% to 80%, 35% to 75%, 35% to70%, 35% to 65%, 35% to 60%, 35% to 55%, 35% to 50%, 35% to 45%, 35% to40%, 40% to 100%, 40% to 95%, 40% to 90%, 40% to 85%, 40% to 80%, 40% to75%, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 55%, 40% to 50%, 40% to45%, 45% to 100%, 45% to 95%, 45% to 90%, 45% to 85%, 45% to 80%, 45% to75%, 45% to 70%, 45% to 65%, 45% to 60%, 45% to 55%, 45% to 50%, 50% to100%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to70%, 50% to 65%, 50% to 60%, 50% to 55%, 55% to 100%, 55% to 95%, 55% to90%, 55% to 85%, 55% to 80%, 55% to 75%, 55% to 70%, 55% to 65%, 55% to60%, 60% to 100%, 60% to 95%, 60% to 90%, 60% to 85%, 60% to 80%, 60% to75%, 60% to 70%, 60% to 65%, 65% to 100%, 65% to 95%, 65% to 90%, 65% to85%, 65% to 80%, 65% to 75%, 65% to 70%, 70% to 100%, 70% to 95%, 70% to90%, 70% to 85%, 70% to 80%, 70% to 75%, 75% to 100%, 75% to 95%, 75% to90%, 75% to 85%, 75% to 80%, 80% to 100%, 80% to 95%, 80% to 90%, 80% to85%, 85% to 100%, 85% to 95%, 85% to 90%, 90% to 100%, 90% to 95%, or95% to 100%.

In some embodiments, the microbial compositions of the presentdisclosure have a water activity of about 0.1, about 0.15, about 0.2,about 0.25, about 0.30, about 0.35, about 0.4, about 0.5, about 0.55,about 0.60, about 0.65, about 0.70, about 0.75, about 0.8, about 0.85,about 0.90, or about 0.95.

In some embodiments, the microbial compositions of the presentdisclosure have a water activity of less than about 0.1, less than about0.15, less than about 0.2, less than about 0.25, less than about 0.30,less than about 0.35, less than about 0.4, less than about 0.5, lessthan about 0.55, less than about 0.60, less than about 0.65, less thanabout 0.70, less than about 0.75, less than about 0.8, less than about0.85, less than about 0.90, or less than about 0.95.

In some embodiments, the microbial compositions of the presentdisclosure have a water activity of less than 0.1, less than 0.15, lessthan 0.2, less than 0.25, less than 0.30, less than 0.35, less than 0.4,less than 0.5, less than 0.55, less than 0.60, less than 0.65, less than0.70, less than 0.75, less than 0.8, less than 0.85, less than 0.90, orless than 0.95.

In some embodiments, the microbial compositions of the presentdisclosure have a water activity of 0.1 to 0.95, 0.1 to 0.90, 0.1 to0.85, 0.1 to 0.8, 0.1 to 0.75, 0.1 to 0.70, 0.1 to 0.65, 0.1 to 0.55,0.1 to 0.50, 0.1 to 0.45, 0.1 to 0.40, 0.1 to 0.35, 0.1 to 0.3, 0.1 to0.25, 0.1 to 0.2, 0.1 to 0.15, 0.15 to 0.95, 0.15 to 0.90, 0.15 to 0.85,0.15 to 0.8, 0.15 to 0.75, 0.15 to 0.70, 0.15 to 0.65, 0.15 to 0.55,0.15 to 0.50, 0.15 to 0.45, 0.15 to 0.40, 0.15 to 0.35, 0.15 to 0.3,0.15 to 0.25, 0.15 to 0.2, 0.2 to 0.95, 0.2 to 0.90, 0.2 to 0.85, 0.2 to0.8, 0.2 to 0.75, 0.2 to 0.70, 0.2 to 0.65, 0.2 to 0.55, 0.2 to 0.50,0.2 to 0.45, 0.2 to 0.40, 0.2 to 0.35, 0.2 to 0.3, 0.2 to 0.25, 0.25 to0.95, 0.25 to 0.90, 0.25 to 0.85, 0.25 to 0.8, 0.25 to 0.75, 0.25 to0.70, 0.25 to 0.65, 0.25 to 0.55, 0.25 to 0.50, 0.25 to 0.45, 0.25 to0.40, 0.25 to 0.35, 0.25 to 0.3, 0.3 to 0.95, 0.3 to 0.90, 0.3 to 0.85,0.3 to 0.8, 0.3 to 0.75, 0.3 to 0.70, 0.3 to 0.65, 0.3 to 0.55, 0.3 to0.50, 0.3 to 0.45, 0.3 to 0.40, 0.3 to 0.35, 0.35 to 0.95, 0.35 to 0.90,0.35 to 0.85, 0.35 to 0.8, 0.35 to 0.75, 0.35 to 0.70, 0.35 to 0.65,0.35 to 0.55, 0.35 to 0.50, 0.35 to 0.45, 0.35 to 0.40, 0.4 to 0.95, 0.4to 0.90, 0.4 to 0.85, 0.4 to 0.8, 0.4 to 0.75, 0.4 to 0.70, 0.4 to 0.65,0.4 to 0.55, 0.4 to 0.50, 0.4 to 0.45, 0.45 to 0.95, 0.45 to 0.90, 0.45to 0.85, 0.45 to 0.8, 0.45 to 0.75, 0.45 to 0.70, 0.45 to 0.65, 0.45 to0.55, 0.45 to 0.50, 0.5 to 0.95, 0.5 to 0.90, 0.5 to 0.85, 0.5 to 0.8,0.5 to 0.75, 0.5 to 0.70, 0.5 to 0.65, 0.5 to 0.55, 0.55 to 0.95, 0.55to 0.90, 0.55 to 0.85, 0.55 to 0.8, 0.55 to 0.75, 0.55 to 0.70, 0.55 to0.65, 0.6 to 0.95, 0.6 to 0.90, 0.6 to 0.85, 0.6 to 0.8, 0.6 to 0.75,0.6 to 0.70, 0.65 to 0.95, 0.65 to 0.90, 0.65 to 0.85, 0.65 to 0.8, 0.65to 0.75, 0.7 to 0.95, 0.7 to 0.90, 0.7 to 0.85, 0.7 to 0.8, 0.75 to0.95, 0.75 to 0.90, 0.75 to 0.85, 0.8 to 0.95, 0.8 to 0.90, 0.8 to 0.85,0.85 to 0.95, 0.85 to 0.90, or 0.9 to 0.95.

Encapsulation Compositions

In some embodiments, the microbes or microbial compositions of thedisclosure are encapsulated in an encapsulating composition. Anencapsulating composition protects the microbes from external stressorsprior to entering the gastrointestinal tract of poultry. In someembodiments, external stressors include thermal and physical stressorsassociated with pelleting and extrusion. In some embodiments, externalstressors include chemicals present in the compositions to whichEncapsulating compositions further create an environment that may bebeneficial to the microbes, such as minimizing the oxidative stresses ofan aerobic environment on anaerobic microbes, preserving the viabilityof the microbes wherein vegetative cells or spores form during thepelleting/extrusion process, etc. See Kalsta et al. (U.S. Pat. No.5,104,662A), Ford (U.S. Pat. No. 5,733,568A), and Mosbach and Nilsson(U.S. Pat. No. 4,647,536A) for encapsulation compositions of microbes,and methods of encapsulating microbes.

In one embodiment, the compositions of the present disclosure exhibit athermal tolerance, which is used interchangeably with heat tolerance andheat resistance. In one embodiment, thermal tolerant compositions of thepresent disclosure are tolerant of the high temperatures associated withfeed manufacturing, mixing of feed and compositions of the presentdisclosure, storage in high heat environments, etc. In one embodiment,thermal tolerant compositions of the present disclosure are resistant toheat-killing and denaturation of the cell wall components and theintracellular environment.

In one embodiments, the encapsulation is a reservoir-type encapsulation.In one embodiment, the encapsulation is a matrix-type encapsulation. Inone embodiment, the encapsulation is a coated matrix-type encapsulation.Burgain et al. (2011. J. Food Eng. 104:467-483) discloses numerousencapsulation embodiments and techniques, all of which are incorporatedby reference.

In some embodiments, the compositions of the present disclosure areencapsulated in one or more of the following: gellan gum, xanthan gum,K-Carrageenan, cellulose acetate phthalate, chitosan, starch, milk fat,whey protein, Ca-alginate, raftilose, raftiline, pectin, saccharide,glucose, maltodextrin, gum arabic, guar, seed flour, alginate, dextrins,dextrans, celluloase, gelatin, gelatin, albumin, casein, gluten, acaciagum, tragacanth, wax, paraffin, stearic acid, monodiglycerides, anddiglycerides. In some embodiments, the compositions of the presentdisclosure are encapsulated by one or more of a polymer, carbohydrate,sugar, plastic, glass, polysaccharide, lipid, wax, oil, fatty acid, orglyceride. In one embodiment, the microbial composition is encapsulatedby a glucose. In one embodiment, the microbial composition isencapsulated by a glucose-containing composition. In one embodiment,formulations of the microbial composition comprise a glucoseencapsulant. In one embodiment, formulations of the microbialcomposition comprise a glucose-encapsulated composition.

In some embodiments, the encapsulation of the compositions of thepresent disclosure is carried out by an extrusion, emulsification,coating, agglomeration, lyophilization, vacuum-drying, or spray-drying.

In one embodiment, the encapsulating composition comprises microcapsuleshaving a multiplicity of liquid cores encapsulated in a solid shellmaterial. For purposes ofthe disclosure, a “multiplicity” of cores isdefined as two or more.

A first category of useful fusible shell materials is that of normallysolid fats, including fats which are already of suitable hardness andanimal or vegetable fats and oils which are hydrogenated until theirmelting points are sufficiently high to serve the purposes of thepresent disclosure. Depending on the desired process and storagetemperatures and the specific material selected, a particular fat can beeither a normally solid or normally liquid material. The terms “normallysolid” and “normally liquid” as used herein refer to the state of amaterial at desired temperatures for storing the resultingmicrocapsules. Since fats and hydrogenated oils do not, strictlyspeaking, have melting points, the term “melting point” is used hereinto describe the minimum temperature at which the fusible materialbecomes sufficiently softened or liquid to be successfully emulsifiedand spray cooled, thus roughly corresponding to the maximum temperatureat which the shell material has sufficient integrity to prevent releaseof the choline cores. “Melting point” is similarly defined herein forother materials which do not have a sharp melting point.

Specific examples of fats and oils useful herein (some of which requirehardening) are as follows: animal oils and fats, such as beef tallow,mutton tallow, lamb tallow, lard or pork fat, fish oil, and sperm oil;vegetable oils, such as canola oil, cottonseed oil, peanut oil, cornoil, olive oil, soybean oil, sunflower oil, safflower oil, coconut oil,palm oil, linseed oil, tung oil, and castor oil; fatty acidmonoglycerides and diglycerides; free fatty acids, such as stearic acid,palmitic acid, and oleic acid; and mixtures thereof. The above listingof oils and fats is not meant to be exhaustive, but only exemplary.

Specific examples of fatty acids include linoleic acid, γ-linoleic acid,dihomo-γ-linolenic acid, arachidonic acid, docosatetraenoic acid,vaccenic acid, nervonic acid, mead acid, erucic acid, gondoic acid,elaidic acid, oleic acid, palitoleic acid, stearidonic acid,eicosapentaenoic acid, valeric acid, caproic acid, enanthic acid,caprylic acid, pelargonic acid, capric acid, undecylic acid, lauricacid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid,margaric acid, stearic acid, nonadecyclic acid, arachidic acid,heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid,pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid,nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid,psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid,heptatriacontanoic acid, and octatriacontanoic acid.

Another category of fusible materials useful as encapsulating shellmaterials is that of waxes. Representative waxes contemplated for useherein are as follows: animal waxes, such as beeswax, lanolin, shellwax, and Chinese insect wax; vegetable waxes, such as carnauba,candelilla, bayberry, and sugar cane; mineral waxes, such as paraffin,microcrystalline petroleum, ozocerite, ceresin, and montan; syntheticwaxes, such as low molecular weight polyolefin (e.g., CARBOWAX), andpolyol ether-esters (e.g., sorbitol); Fischer-Tropsch process syntheticwaxes; and mixtures thereof. Water-soluble waxes, such as CARBOWAX andsorbitol, are not contemplated herein if the core is aqueous.

Still other fusible compounds useful herein are fusible natural resins,such as rosin, balsam, shellac, and mixtures thereof.

Various adjunct materials are contemplated for incorporation in fusiblematerials according to the present disclosure. For example,antioxidants, light stabilizers, dyes and lakes, flavors, essentialoils, anti-caking agents, fillers, pH stabilizers, sugars(monosaccharides, disaccharides, trisaccharides, and polysaccharides)and the like can be incorporated in the fusible material in amountswhich do not diminish its utility for the present disclosure.

The core material contemplated herein constitutes from about 0.1% toabout 50%, about 1% to about 35%. or about 5% to about 30% by weight ofthe microcapsules. In some embodiments, the core material contemplatedherein constitutes no more than about 30% by weight of themicrocapsules. In some embodiments, the core material contemplatedherein constitutes about 5% by weight of the microcapsules. The corematerial is contemplated as either a liquid or solid at contemplatedstorage temperatures of the microcapsules.

The cores may include other additives well-known in the pharmaceuticalart, including edible sugars, such as sucrose, glucose, maltose,fructose, lactose, cellobiose, monosaccharides, disaccharides,trisaccharides, and polysaccharides, and mixtures thereof; artificialsweeteners, such as aspartame, saccharin, cyclamate salts, and mixturesthereof; edible acids, such as acetic acid (vinegar), citric acid,ascorbic acid, tartaric acid, and mixtures thereof; edible starches,such as corn starch; hydrolyzed vegetable protein; water-solublevitamins, such as Vitamin C; water-soluble medicaments; water-solublenutritional materials, such as ferrous sulfate; flavors; salts;monosodium glutamate; antimicrobial agents, such as sorbic acid;antimycotic agents, such as potassium sorbate, sorbic acid, sodiumbenzoate, and benzoic acid; food grade pigments and dyes; and mixturesthereof. Other potentially useful supplemental core materials will beapparent to those of ordinary skill in the art.

Emulsifying agents may be employed to assist in the formation of stableemulsions. Representative emulsifying agents include glycerylmonostearate, polysorbate esters, ethoxylated mono- and diglycerides,and mixtures thereof.

For ease of processing, and particularly to enable the successfulformation of a reasonably stable emulsion, the viscosities of the corematerial and the shell material should be similar at the temperature atwhich the emulsion is formed. In particular, the ratio of the viscosityof the shell to the viscosity of the core, expressed in centipoise orcomparable units, and both measured at the temperature of the emulsion,should be from about 22:1 to about 1:1, desirably from about 8:1 toabout 1:1, and preferably from about 3:1 to about 1:1. A ratio of 1:1would be ideal, but a viscosity ratio within the recited ranges isuseful.

Encapsulating compositions are not limited to microcapsule compositionsas disclosed above. In some embodiments encapsulating compositionsencapsulate the microbial compositions in an adhesive polymer that canbe natural or synthetic without toxic effect. In some embodiments, theencapsulating composition may be a matrix selected from sugar matrix,gelatin matrix, polymer matrix, silica matrix, starch matrix, foammatrix, etc. In some embodiments, the encapsulating composition may beselected from polyvinyl acetates; polyvinyl acetate copolymers; ethylenevinyl acetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcoholcopolymers; celluloses, including ethylcelluloses, methylcelluloses,hydroxymethylcelluloses, hydroxypropylcelluloses andcarboxymethylcellulose; polyvinylpyrolidones; polysaccharides, includingstarch, modified starch, dextrins, maltodextrins, alginate andchitosans; monosaccharides; fats; fatty acids, including oils; proteins,including gelatin and zeins; gum arabics; shellacs; vinylidene chlorideand vinylidene chloride copolymers; calcium lignosulfonates; acryliccopolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymersand copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers;and polychloroprene.

In some embodiments, the microbial composition or a subcomponent thereofis encapsulated in a solid glass matrix or a flexible glass matrix(rubber matrix) comprising one or more polysaccharides, one or moresaccharides, and/or one or more sugar alcohols. In some embodiments, thematrix comprises a monosaccharide or a disaccharide. In someembodiments, the disaccharide may be selected from sucrose, maltose,lactose, lactulose, trehalose, cellobiose, and chitobiose. In someembodiments, the polysaccharides, saccharides, and/or sugar alcohols areadded to the microbial composition or a subcomponent thereofexogenously. In some embodiments, the matrix is an amorphous matrix. Insome embodiments, the microbial composition or a subcompenent thereof isvitrified. In some embodiments, the microbial composition or asubcompenent thereof is desiccated. In some embodiments, the microbialcomposition or a subcompenent thereof is lyophilized. In someembodiments, the microbial composition or a subcompenent thereof isspray dried. In some embodiments, the microbial composition or asubcompenent thereof is spray congealed. See Harel and Kohavi-Beck (U.S.Pat. No. 8,097,245).

In some embodiments, the encapsulating shell of the present disclosurecan be up to 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540μm, 550 μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630μm, 640 μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720μm, 730 μm, 740 μm, 750 μm, 760 μm, 770 μm, 780 μm, 790 μm, 800 μm, 810μm, 820 μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 880 μm, 890 μm, 900μm, 910 μm, 920 μm, 930 μm, 940 μm, 950 μm, 960 μm, 970 μm, 980 μm, 990μm, 1000 μm, 1010 μm, 1020 μm, 1030 μm, 1040 μm, 1050 μm, 1060 μm, 1070μm, 1080 μm, 1090 μm, 1100 μm, 1110 μm, 1120 μm, 1130 μm, 1140 μm, 1150μm, 1160 μm, 1170 μm, 1180 μm, 1190 μm, 1200 μm, 1210 μm, 1220 μm, 1230μm, 1240 μm, 1250 μm, 1260 μm, 1270 μm, 1280 μm, 1290 μm, 1300 μm, 1310μm, 1320 μm, 1330 μm, 1340 μm, 1350 μm, 1360 μm, 1370 μm, 1380 μm, 1390μm, 1400 μm, 1410 μm, 1420 μm, 1430 μm, 1440 μm, 1450 μm, 1460 μm, 1470μm, 1480 μm, 1490 μm, 1500 μm, 1510 μm, 1520 μm, 1530 μm, 1540 μm, 1550μm, 1560 μm, 1570 μm, 1580 μm, 1590 μm, 1600 μm, 1610 μm, 1620 μm, 1630μm, 1640 μm, 1650 μm, 1660 μm, 1670 μm, 1680 μm, 1690 μm, 1700 μm, 1710μm, 1720 μm, 1730 μm, 1740 μm, 1750 μm, 1760 μm, 1770 μm, 1780 μm, 1790μm, 1800 μm, 1810 μm, 1820 μm, 1830 μm, 1840 μm, 1850 μm, 1860 μm, 1870μm, 1880 μm, 1890 μm, 1900 μm, 1910 μm, 1920 μm, 1930 μm, 1940 μm, 1950μm, 1960 μm, 1970 μm, 1980 μm, 1990 μm, 2000 μm, 2010 μm, 2020 μm, 2030μm, 2040 μm, 2050 μm, 2060 μm, 2070 μm, 2080 μm, 2090 μm, 2100 μm, 2110μm, 2120 μm, 2130 μm, 2140 μm, 2150 μm, 2160 μm, 2170 μm, 2180 μm, 2190μm, 2200 μm, 2210 μm, 2220 μm, 2230 μm, 2240 μm, 2250 μm, 2260 μm, 2270μm, 2280 μm, 2290 μm, 2300 μm, 2310 μm, 2320 μm, 2330 μm, 2340 μm, 2350μm, 2360 μm, 2370 μm, 2380 μm, 2390 μm, 2400 μm, 2410 μm, 2420 μm, 2430μm, 2440 μm, 2450 μm, 2460 μm, 2470 μm, 2480 μm, 2490 μm, 2500 μm, 2510μm, 2520 μm, 2530 μm, 2540 μm, 2550 μm, 2560 μm, 2570 μm, 2580 μm, 2590μm, 2600 μm, 2610 μm, 2620 μm, 2630 μm, 2640 μm, 2650 μm, 2660 μm, 2670μm, 2680 μm, 2690 μm, 2700 μm, 2710 μm, 2720 μm, 2730 μm, 2740 μm, 2750μm, 2760 μm, 2770 μm, 2780 μm, 2790 μm, 2800 μm, 2810 μm, 2820 μm, 2830μm, 2840 μm, 2850 μm, 2860 μm, 2870 μm, 2880 μm, 2890 μm, 2900 μm, 2910μm, 2920 μm, 2930 μm, 2940 μm, 2950 μm, 2960 μm, 2970 μm, 2980 μm, 2990μm, or 3000 μm thick.

Animal Feed

In some embodiments, compositions of the present disclosure are mixedwith animal feed. In some embodiments, animal feed may be present invarious forms such as pellets, capsules, granulated, powdered, mash,liquid, or semi-liquid. In some embodiments, compositions of the presentdisclosure are mixed with pre-pelleted feed. In some embodiments,compositions of the present disclosure are mixed with pelleted feed. Insome embodiments, compositions of the present disclosure are mixed withfeed after it has been pelleted.

In some embodiments, compositions of the present disclosure are mixedinto the premix or mash at the feed mill, alone as a standalone premix,and/or alongside other feed additives such as MONENSIN, vitamins, etc.In one embodiment, the compositions of the present disclosure are mixedinto or onto the feed at the feed mill. In another embodiment,compositions of the present disclosure are mixed into the feed itself

In some embodiments, feed of the present disclosure may be supplementedwith water, premix or premixes, forage, fodder, beans (e.g., whole,cracked, or ground), grains (e.g., whole, cracked, or ground), bean- orgrain-based oils, bean- or grain-based meals, bean- or grain-basedhaylage or silage, bean- or grain-based syrups, fatty acids, sugaralcohols (e.g., polyhydric alcohols), commercially available formulafeeds, oyster shells and those of other bivalves, and mixtures thereof.

In some embodiments, forage encompasses hay, haylage, and silage. Insome embodiments, hays include grass hays (e.g., sudangrass,orchardgrass, or the like), alfalfa hay, and clover hay. In someembodiments, haylages include grass haylages, sorghum haylage, andalfalfa haylage. In some embodiments, silages include maize, oat, wheat,alfalfa, clover, and the like.

In some embodiments, premix or premixes may be utilized in the feed.Premixes may comprise micro-ingredients such as vitamins, minerals,amino acids; chemical preservatives; pharmaceutical compositions such asantibiotics and other medicaments; fermentation products, and otheringredients. In some embodiments, premixes are blended into the feed.

In some embodiments, the feed may include feed concentrates such assoybean hulls, soybean oils, sugar beet pulp, molasses, high proteinsoybean meal, ground corn, shelled corn, wheat midds, distiller grain,cottonseed hulls, and grease. See Anderson et al. (U.S. Pat. No.3,484,243), Iritani et al. (U.S. Pat. No. 6,090,416), Axelrod et al.(U.S. Publication US20060127530A1), and Katsumi et al. (U.S. Pat. No.5,741,508) for animal feed and animal feed supplements capable of use inthe present compositions and methods.

In some embodiments, feed occurs as a compound, which includes, in amixed composition capable of meeting the basic dietary needs, the feeditself, vitamins, minerals, amino acids, and other necessary components.Compound feed may further comprise premixes.

In some embodiments, microbial compositions of the present disclosuremay be mixed with animal feed, premix, and/or compound feed. Individualcomponents of the animal feed may be mixed with the microbialcompositions prior to feeding to poultry. The microbial compositions ofthe present disclosure may be applied into or on a premix, into or on afeed, and/or into or on a compound feed.

Administration of Microbial Compositions

In some embodiments, the microbial compositions of the presentdisclosure are administered to poultry via the oral route. In someembodiments the microbial compositions are administered via a directinjection route into the gastrointestinal tract. In further embodiments,the direct injection administration delivers the microbial compositionsdirectly to one or more of the crop, gizzard, cecum, small intestine,and large intestine. FIG. 12 and FIG. 13 provide a detailed anatomicalview of the gastrointestinal tract of a chicken. In some embodiments,the microbial compositions of the present disclosure are administered toanimals through the cloaca. In further embodiments, cloacaladministration is in the form of an inserted suppository.

In some embodiments, the microbial compositions are administered throughdrinking water, spraying on litter in which the animal is in contactwith, mixing with medications or vaccines, and gavage. In someembodiments, the microbial compositions are sprayed directly on theanimal, wherein the animal ingests the composition having been sprayedon the animal. In some embodiments, the microbial compositions aresprayed on and/or sprayed in feed, and the feed is administered to theanimal. In further embodiments, the animal ingests the compositionthrough the preening of feathers that have come into contact with thesprayed composition.

In some embodiments, the microbial compositions of the presentdisclosure are administered to poultry on day 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, or 31 post-hatching. In some embodiments, the microbialcompositions are administered to the exterior surface of an egg as aliquid, semi-liquid, or solid on day 22, 21, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 pre-hatching. In someembodiments, the microbial compositions of the present disclosure areadministered to poultry in multiple dosing sessions in week(s) 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, and/or 30 week(s) post-hatching. In someembodiments, the microbial compositions are administered immediatelyafter hatching. In some embodiments, the microbial compositions areadministered into the egg (e.g., injection) by itself or administeredalong with other products such as vaccines.

In some embodiments, the microbial composition is administered in a dosecomprise a total of, or at least, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml,18 ml, 19 ml, 20 ml, 21 ml, 22 ml, 23 ml, 24 ml, 25 ml, 26 ml, 27 ml, 28ml, 29 ml, 30 ml, 31 ml, 32 ml, 33 ml, 34 ml, 35 ml, 36 ml, 37 ml, 38ml, 39 ml, 40 ml, 41 m, 42 ml, 43 ml, 44 ml, 45 ml, 46 ml, 47 ml, 48 ml,49 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, or 1,000 ml.

In some embodiments, the microbial composition is administered in a dosecomprising a total of, or at least, 10¹⁸, 1017, 1016, 1015, 1014, 1013,1012, 1011, 1010, 10⁹, 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, or 10² microbialcells.

In some embodiments, the microbial compositions are mixed with feed, andthe administration occurs through the ingestion of the microbialcompositions along with the feed. In some embodiments, the dose of themicrobial composition is administered such that there exists 10² to10¹², 10³ to 10¹², 10⁴ to 10¹², 10⁵ to 10¹², 10⁶ to 10¹², 10⁷ to 10¹²,10⁸ to 10¹², 10⁹ to 10¹², 10¹⁰ to 10¹², 10¹¹ to 10¹², 10² to 10¹¹, 10³to 10¹¹, 10⁴ to 10¹¹, 10⁵ to 10¹¹, 10⁶ to 10¹¹, 10⁷ to 10¹¹, 10⁸ to10¹¹, 10⁹ to 10¹¹, 10¹⁰ to 10¹¹, 10² to 10¹⁰, 10³ to 10¹⁰, 10⁴ to 10¹⁰,10⁵ to 10¹⁰, 10⁶ to 10¹⁰, 10⁷ to 10¹⁰, 10⁸ to 10¹⁰, 10⁹ to 10¹⁰, 10² to10⁹, 10³ to 10⁹, 10⁴ to 10⁹, 10⁵ to 10⁹, 10⁶ to 10⁹, 10⁷ to 10⁹, 10⁸ to10⁹, 10² to 10⁸, 10³ to 10⁸, 10⁴ to 10⁸, 10⁵ to 10⁸, 10⁶ to 10⁸, 10⁷ to10⁸, 10² to 10⁷, 10³ to 10⁷, 10⁴ to 10⁷, 10⁵ to 10⁷, 10⁶ to 10⁷, 10² to10⁶, 10³ to 10⁶, 10⁴ to 10⁶, 10⁵ to 10⁶, 10² to 10⁵, 10³ to 10⁵, 10⁴ to10⁵, 10² to 10⁴, 10³ to 10⁴, 10² to 10³, 10¹², 10¹¹, 10¹⁰, 10⁹, 10⁸,10⁷, 10⁶, 10⁵, 10⁴, 10³, or 10² total microbial cells per gram ormilliliter of the composition.

In some embodiments, the administered dose of the microbial compositioncomprises 10² to 10¹⁸, 10³ to 10¹⁸, 10⁴ to 10¹⁸, 10⁵ to 10¹⁸, 10⁶ to10¹⁸, 10⁷ to 10¹⁸, 10⁸ to 10¹⁸, 10⁹ to 10¹⁸, 10¹⁰ to 10¹⁸, 10¹¹ to 10¹⁸,10¹² to 10¹⁸, 10¹³ to 10¹⁸, 10¹⁴ to 10¹⁸, 10¹⁵ to 10¹⁸, 10¹⁶ to 10¹⁸,10¹⁷ to 10¹⁸, 10² to 10¹², 10³ to 10¹², 10⁴ to 10¹², 10⁵ to 10¹², 10⁶ to10¹², 10 to 10¹², 10⁸ to 10¹², 10⁹ to 10¹², 10¹⁰ to 10¹², 10¹¹ to 10¹²,10² to 10¹¹, 10³ to 10¹¹, 10⁴ to 10¹¹, 10⁵ to 10¹¹, 10⁶ to 10¹¹, 10⁷ to10¹¹, 10⁸ to 10¹¹, 10⁹ to 10¹¹, 10¹⁰ to 10¹¹, 10² to 10¹⁰, 10³ to 10¹⁰,10⁴ to 10¹⁰, 10⁵ to 10¹⁰, 10⁶ to 10¹⁰, 10⁷ to 10¹⁰, 10⁸ to 10¹⁰, 10⁹ to10¹⁰, 10² to 10⁹, 10³ to 10⁹, 10⁴ to 10⁹, 10⁵ to 10⁹, 10⁶ to 10⁹, 10⁷ to10⁹, 10⁸ to 10⁹, 10² to 10⁸, 10³ to 10⁸, 10⁴ to 10⁸, 10⁵ to 10⁸, 10⁶ to10⁸, 10⁷ to 10⁸, 10² to 10⁷, 10³ to 10⁷, 10⁴ to 10⁷, 10⁵ to 10⁷, 10⁶ to10⁷, 10² to 10⁶, 10³ to 10⁶, 10⁴ to 10⁶, 10⁵ to 10⁶, 10² to 10⁵, 10³ to10⁵, 10⁴ to 10⁵, 10² to 10⁴, 10³ to 10⁴, 10² to 10³, 10¹⁸, 10¹⁷, 10¹⁶,10¹⁵, 10¹⁴, 10¹³, 10¹², 10¹¹, 10¹⁰, 10⁹, 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³,or 10² total microbial cells.

In some embodiments, the composition is administered 1 or more times perday. In some aspects, the composition is administered with food eachtime the animal is fed. In some embodiments, the composition isadministered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10,4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7,5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day.

In some embodiments, the microbial composition is administered 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10,2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9,3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7,4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times per week.

In some embodiments, the microbial composition is administered 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10,2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9,3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7,4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times per month.

In some embodiments, the microbial composition is administered 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10,2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9,3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7,4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times per year.

In some embodiments, the feed can be uniformly coated with one or morelayers of the microbes and/or microbial compositions disclosed herein,using conventional methods of mixing, spraying, or a combination thereofthrough the use of treatment application equipment that is specificallydesigned and manufactured to accurately, safely, and efficiently applycoatings. Such equipment uses various types of coating technology suchas rotary coaters, drum coaters, fluidized bed techniques, spouted beds,rotary mists, or a combination thereof. Liquid treatments such as thoseof the present disclosure can be applied via either a spinning“atomizer” disk or a spray nozzle, which evenly distributes themicrobial composition onto the feed as it moves though the spraypattern. In some aspects, the feed is then mixed or tumbled for anadditional period of time to achieve additional treatment distributionand drying.

In some embodiments, the feed coats of the present disclosure can be upto 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540 μm, 550μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630 μm, 640μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720 μm, 730μm, 740 μm, 750 μm, 760 μm, 770 μm, 780 μm, 790 μm, 800 μm, 810 μm, 820μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 880 μm, 890 μm, 900 μm, 910μm, 920 μm, 930 μm, 940 μm, 950 μm, 960 μm, 970 μm, 980 μm, 990 μm, 1000μm, 1010 μm, 1020 μm, 1030 μm, 1040 μm, 1050 μm, 1060 μm, 1070 μm, 1080μm, 1090 μm, 1100 μm, 1110 μm, 1120 μm, 1130 μm, 1140 μm, 1150 μm, 1160μm, 1170 μm, 1180 μm, 1190 μm, 1200 μm, 1210 μm, 1220 μm, 1230 μm, 1240μm, 1250 μm, 1260 μm, 1270 μm, 1280 μm, 1290 μm, 1300 μm, 1310 μm, 1320μm, 1330 μm, 1340 μm, 1350 μm, 1360 μm, 1370 μm, 1380 μm, 1390 μm, 1400μm, 1410 μm, 1420 μm, 1430 μm, 1440 μm, 1450 μm, 1460 μm, 1470 μm, 1480μm, 1490 μm, 1500 μm, 1510 μm, 1520 μm, 1530 μm, 1540 μm, 1550 μm, 1560μm, 1570 μm, 1580 μm, 1590 μm, 1600 μm, 1610 μm, 1620 μm, 1630 μm, 1640μm, 1650 μm, 1660 μm, 1670 μm, 1680 μm, 1690 μm, 1700 μm, 1710 μm, 1720μm, 1730 μm, 1740 μm, 1750 μm, 1760 μm, 1770 μm, 1780 μm, 1790 μm, 1800μm, 1810 μm, 1820 μm, 1830 μm, 1840 μm, 1850 μm, 1860 μm, 1870 μm, 1880μm, 1890 μm, 1900 μm, 1910 μm, 1920 μm, 1930 μm, 1940 μm, 1950 μm, 1960μm, 1970 μm, 1980 μm, 1990 μm, 2000 μm, 2010 μm, 2020 μm, 2030 μm, 2040μm, 2050 μm, 2060 μm, 2070 μm, 2080 μm, 2090 μm, 2100 μm, 2110 μm, 2120μm, 2130 μm, 2140 μm, 2150 μm, 2160 μm, 2170 μm, 2180 μm, 2190 μm, 2200μm, 2210 μm, 2220 μm, 2230 μm, 2240 μm, 2250 μm, 2260 μm, 2270 μm, 2280μm, 2290 μm, 2300 μm, 2310 μm, 2320 μm, 2330 μm, 2340 μm, 2350 μm, 2360μm, 2370 μm, 2380 μm, 2390 μm, 2400 μm, 2410 μm, 2420 μm, 2430 μm, 2440μm, 2450 μm, 2460 μm, 2470 μm, 2480 μm, 2490 μm, 2500 μm, 2510 μm, 2520μm, 2530 μm, 2540 μm, 2550 μm, 2560 μm, 2570 μm, 2580 μm, 2590 μm, 2600μm, 2610 μm, 2620 μm, 2630 μm, 2640 μm, 2650 μm, 2660 μm, 2670 μm, 2680μm, 2690 μm, 2700 μm, 2710 μm, 2720 μm, 2730 μm, 2740 μm, 2750 μm, 2760μm, 2770 μm, 2780 μm, 2790 μm, 2800 μm, 2810 μm, 2820 μm, 2830 μm, 2840μm, 2850 μm, 2860 μm, 2870 μm, 2880 μm, 2890 μm, 2900 μm, 2910 μm, 2920μm, 2930 μm, 2940 μm, 2950 μm, 2960 μm, 2970 μm, 2980 μm, 2990 μm, or3000 μm thick.

In some embodiments, the microbial cells can be coated freely onto anynumber of compositions or they can be formulated in a liquid or solidcomposition before being coated onto a composition. For example, a solidcomposition comprising the microorganisms can be prepared by mixing asolid carrier with a suspension of the spores until the solid carriersare impregnated with the spore or cell suspension. This mixture can thenbe dried to obtain the desired particles.

In some other embodiments, it is contemplated that the solid or liquidmicrobial compositions of the present disclosure further containfunctional agents e.g., activated carbon, minerals, vitamins, and otheragents capable of improving the quality of the products or a combinationthereof.

Methods of coating and compositions in use of said methods that areknown in the art can be particularly useful when they are modified bythe addition of one of the embodiments of the present disclosure. Suchcoating methods and apparatus for their application are disclosed in,for example: U.S. Pat. Nos. 8,097,245 and 7,998,502; and PCT Pat. App.Publication Nos. WO 2008/076975, WO 2010/138522, WO2011/094469, WO2010/111347, and WO 2010/111565, each of which is incorporated byreference herein.

In some embodiments, the microbes or microbial ensembles of the presentdisclosure exhibit a synergistic effect, on one or more of the traitsdescribed herein, in the presence of one or more of the microbes ormicrobial ensembles coming into contact with one another. Thesynergistic effect obtained by the taught methods can be quantified, forexample, according to Colby's formula (i.e., (E)=X+Y−(X*Y/100)). SeeColby, R. S., “Calculating Synergistic and Antagonistic Responses ofHerbicide Combinations,” 1967. Weeds. Vol. 15, pp. 20-22, incorporatedherein by reference in its entirety. Thus, “synergistic” is intended toreflect an outcome/parameter/effect that has been increased by more thanan additive amount.

In some embodiments, the microbes or microbial ensembles of the presentdisclosure may be administered via drench. In one embodiment, the drenchis an oral drench. A drench administration comprises utilizing a drenchkit/applicator/syringe that injects/releases a liquid comprising themicrobes or microbial ensembles into the buccal cavity and/or esophagasof the animal.

In some embodiments, the microbes or microbial ensembles of the presentdisclosure may be administered in a time-released fashion. Thecomposition may be coated in a chemical composition, or may be containedin a mechanical device or capsule that releases the microbes ormicrobial ensembles over a period of time instead all at once. In oneembodiment, the microbes or microbial ensembles are administered to ananimal in a time-release capsule. In one embodiment, the composition maybe coated in a chemical composition, or may be contained in a mechanicaldevice or capsule that releases the microbes or microbial ensembles allat once a period of time hours post ingestion.

In some embodiments, the microbes or microbial ensembles areadministered in a time-released fashion between 1 to 5, 1 to 10, 1 to15, 1 to 20, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to90, 1 to 95, or 1 to 100 hours.

In some embodiments, the microbes or microbial ensembles areadministered in a time-released fashion between 1 to 2, 1 to 3, 1 to 4,1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to29, or 1 to 30 days.

Microorganisms

As used herein the term “microorganism” should be taken broadly. Itincludes, but is not limited to, the two prokaryotic domains, Bacteriaand Archaea, as well as eukaryotic fungi, protists, and viruses.

By way of example, the microorganisms may include species of the generaof: Lactobacillus, Clostridium, Faecalibacter, Hydrogenoanaerobacterium,Acrocarpospora, Bacillus, Subdoligranulum, Leuconostoc, Lachnospira,Anaerofilum, Microbacterium, Verrucosispora, Blautia, Pseudomonas,Sporobacter, Corynebacterium Streptococcus, Paracoccus, Celulosilyticum,Ruminococcus, Bacteroides, Filobasidium, Gibberella, Alatospora, Pichia,and Candida. In some embodiments, the microorganisms may include speciesof any general disclosed herein.

In certain embodiments, the microorganism is unculturable. This shouldbe taken to mean that the microorganism is not known to be culturable oris difficult to culture using methods known to one skilled in the art.

In one embodiment, the microbes are obtained from animals (e.g.,mammals, reptiles, birds, and the like), soil (e.g., rhizosphere), air,water (e.g., marine, freshwater, wastewater sludge), sediment, oil,plants (e.g., roots, leaves, stems), agricultural products, and extremeenvironments (e.g., acid mine drainage or hydrothermal systems). In afurther embodiment, microbes obtained from marine or freshwaterenvironments such as an ocean, river, or lake. In a further embodiment,the microbes can be from the surface of the body of water, or any depthof the body of water (e.g., a deep sea sample).

The microorganisms of the disclosure may be isolated in substantiallypure or mixed cultures. They may be concentrated, diluted, or providedin the natural concentrations in which they are found in the sourcematerial. For example, microorganisms from saline sediments may beisolated for use in this disclosure by suspending the sediment in freshwater and allowing the sediment to fall to the bottom. The watercontaining the bulk of the microorganisms may be removed by decantationafter a suitable period of settling and either administered to the GItract of poultry, or concentrated by filtering or centrifugation,diluted to an appropriate concentration and administered to the GI tractof poultry with the bulk of the salt removed. By way of further example,microorganisms from mineralized or toxic sources may be similarlytreated to recover the microbes for application to poultry to minimizethe potential for damage to the animal.

In another embodiment, the microorganisms are used in a crude form, inwhich they are not isolated from the source material in which theynaturally reside. For example, the microorganisms are provided incombination with the source material in which they reside; for example,fecal matter or other composition found in the gastrointestinal tract.In this embodiment, the source material may include one or more speciesof microorganisms.

In some embodiments, a mixed population of microorganisms is used in themethods of the disclosure.

In embodiments of the disclosure where the microorganisms are isolatedfrom a source material (for example, the material in which theynaturally reside), any one or a combination of a number of standardtechniques which will be readily known to skilled persons may be used.However, by way of example, these in general employ processes by which asolid or liquid culture of a single microorganism can be obtained in asubstantially pure form, usually by physical separation on the surfaceof a solid microbial growth medium or by volumetric dilutive isolationinto a liquid microbial growth medium. These processes may includeisolation from dry material, liquid suspension, slurries or homogenatesin which the material is spread in a thin layer over an appropriatesolid gel growth medium, or serial dilutions of the material made into asterile medium and inoculated into liquid or solid culture media.

Whilst not essential, in one embodiment, the material containing themicroorganisms may be pre-treated prior to the isolation process inorder to either multiply all microorganisms in the material.Microorganisms can then be isolated from the enriched materials asdisclosed above.

In certain embodiments, as mentioned herein before, the microorganism(s)may be used in crude form and need not be isolated from an animal or amedia. For example, feces, or growth media which includes themicroorganisms identified to be of benefit to increased feed efficiencymay be obtained and used as a crude source of microorganisms for thenext round of the method or as a crude source of microorganisms at theconclusion of the method. For example, fresh feces could be obtained andoptionally processed.

Microbiome Shift and Abundance of Microbes

In some embodiments, the microbiome of poultry, including the gutmicrobiome (crop, gizzard, cecum, small intestine, and large intestine)comprises a diverse arrive of microbes with a wide variety of metaboliccapabilities. The microbiome is influenced by a range of factorsincluding diet, variations in animal metabolism, and breed, amongothers. Most poultry diets are plant-based and rich in complexpolysaccharides that enrich the gastrointestinal microbial community formicrobes capable of breaking down specific polymeric components in thediet such as cellulose, hemicellulose, lignin, etc. The end products ofprimary degradation sustain a chain of microbes that ultimately producea range of organic acids together with hydrogen and carbon dioxide.Because of the complex and interlinked nature of the microbiome,changing the diet and thus substrates for primary degradation may have acascading effect on gut microbial metabolism, with changes in both theorganic acid profiles and the methane levels produced, thus impactingthe quality and quantity of animal production and or the productsproduced by the animal. See Menezes et al. (2011. FEMS Microbiol. Ecol.78(2):256-265.)

In some aspects, the present disclosure is drawn to administeringmicrobial compositions described herein to modulate or shift themicrobiome of poultry.

In some embodiments, the microbiome is shifted through theadministration of one or more microbes to the gastrointestinal tract. Infurther embodiments, the one or more microbes are those selected fromTable 1 and/or Table 3. In some embodiments, the microbiome shift ormodulation includes a decrease or loss of specific microbes that werepresent prior to the administration of one or more microbes of thepresent disclosure. In some embodiments, the microbiome shift ormodulation includes an increase in microbes that were present prior tothe administration of one or more microbes of the present disclosure. Insome embodiments, the microbiome shift or modulation includes a gain ofone or more microbes that were not present prior to the administrationof one or more microbes of the present disclosure. In a furtherembodiment, the gain of one or more microbes is a microbe that was notspecifically included in the administered microbial ensemble.

In some embodiments, the administration of microbes of the presentdisclosure results in a sustained modulation of the microbiome such thatthe administered microbes are present in the microbiome for a period ofat least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10,8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

In some embodiments, the administration of microbes of the presentdisclosure results in a sustained modulation of the microbiome such thatthe administered microbes are present in the microbiome for a period ofat least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10,8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.

In some embodiments, the administration of microbes of the presentdisclosure results in a sustained modulation of the microbiome such thatthe administered microbes are present in the microbiome for a period ofat least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10,8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

In some embodiments, the presence of the administered microbes aredetected by sampling the gastrointestinal tract and using primers toamplify the 16S or 18S rDNA sequences, or the ITS rDNA sequences of theadministered microbes. In some embodiments, the administered microbesare one or more of those selected from Table 1 and/or Table 3, and thecorresponding rDNA sequences are those selected from SEQ ID NOs:1-387.

In some embodiments, the microbiome of a bird is measured by amplifyingpolynucleotides collected from gastrointestinal samples, wherein thepolynucleotides may be 16S or 18S rDNA fragments, or ITS rDNA fragmentsof microbial rDNA. In one embodiment, the microbiome is fingerprinted bya method of denaturing gradient gel electrophoresis (DGGE) wherein theamplified rDNA fragments are sorted by where they denature, and form aunique banding pattern in a gel that may be used for comparing themicrobiome of the same bird over time or the microbiomes of multiplebirds. In another embodiment, the microbiome is fingerprinted by amethod of terminal restriction fragment length polymorphism (T-RFLP),wherein labelled PCR fragments are digested using a restriction enzymeand then sorted by size. In a further embodiment, the data collectedfrom the T-RFLP method is evaluated by nonmetric multidimensionalscaling (nMDS) ordination and PERMANOVA statistics identify differencesin microbiomes, thus allowing for the identification and measurement ofshifts in the microbiome. See also Shanks et al. (2011. Appl. Environ.Microbiol. 77(9):2992-3001), Petri et al. (2013. PLOS one.8(12):e83424), and Menezes et al. (2011. FEMS Microbiol. Ecol.78(2):256-265.)

In some embodiments, the administration of microbes of the presentdisclosure results in a modulation or shift of the microbiome whichfurther results in a desired phenotype or improved trait.

MIC Scoring

According to the methods provided herein, a sample is processed todetect the presence of one or more microorganism types in the sample(FIG. 1, 1001; FIG. 2, 2001). The absolute number of one or moremicroorganism organism type in the sample is determined (FIG. 1, 1002;FIG. 2, 2002). The determination of the presence of the one or moreorganism types and the absolute number of at least one organism type canbe conducted in parallel or serially. For example, in the case of asample comprising a microbial community comprising bacteria (i.e., onemicroorganism type) and fungi (i.e., a second microorganism type), theuser in one embodiment detects the presence of one or both of theorganism types in the sample (FIG. 1, 1001; FIG. 2, 2001). The user, ina further embodiment, determines the absolute number of at least oneorganism type in the sample—in the case of this example, the number ofbacteria, fungi or combination thereof, in the sample (FIG. 1, 1002;FIG. 2, 2002).

In one embodiment, the sample, or a portion thereof is subjected to flowcytometry (FC) analysis to detect the presence and/or number of one ormore microorganism types (FIG. 1, 1001, 1002; FIG. 2, 2001, 2002). Inone flow cytometer embodiment, individual microbial cells pass throughan illumination zone, at a rate of at least about 300*s⁻¹, or at leastabout 500*s⁻¹, or at least about 1000*s⁻¹. However, one of ordinaryskill in the art will recognize that this rate can vary depending on thetype of instrument is employed. Detectors which are gated electronicallymeasure the magnitude of a pulse representing the extent of lightscattered. The magnitudes of these pulses are sorted electronically into“bins” or “channels,” permitting the display of histograms of the numberof cells possessing a certain quantitative property (e.g., cell stainingproperty, diameter, cell membrane) versus the channel number. Suchanalysis allows for the determination of the number of cells in each“bin” which in embodiments described herein is an “microorganism type”bin, e.g., a bacteria, fungi, nematode, protozoan, archaea, algae,dinoflagellate, virus, viroid, etc.

In one embodiment, a sample is stained with one or more fluorescent dyeswherein a fluorescent dye is specific to a particular microorganismtype, to enable detection via a flow cytometer or some other detectionand quantification method that harnesses fluorescence, such asfluorescence microscopy. The method can provide quantification of thenumber of cells and/or cell volume of a given organism type in a sample.In a further embodiment, as described herein, flow cytometry isharnessed to determine the presence and quantity of a unique firstmarker and/or unique second marker of the organism type, such as enzymeexpression, cell surface protein expression, etc. Two- or three-variablehistograms or contour plots of, for example, light scattering versusfluorescence from a cell membrane stain (versus fluorescence from aprotein stain or DNA stain) may also be generated, and thus animpression may be gained of the distribution of a variety of propertiesof interest among the cells in the population as a whole. A number ofdisplays of such multiparameter flow cytometric data are in common useand are amenable for use with the methods described herein.

In one embodiment of processing the sample to detect the presence andnumber of one or more microorganism types, a microscopy assay isemployed (FIG. 1, 1001, 1002). In one embodiment, the microscopy isoptical microscopy, where visible light and a system of lenses are usedto magnify images of small samples. Digital images can be captured by acharge-couple device (CCD) camera. Other microscopic techniques include,but are not limited to, scanning electron microscopy and transmissionelectron microscopy. Microorganism types are visualized and quantifiedaccording to the aspects provided herein.

In another embodiment of in order to detect the presence and number ofone or more microorganism types, the sample, or a portion thereof issubjected to fluorescence microscopy. Different fluorescent dyes can beused to directly stain cells in samples and to quantify total cellcounts using an epifluorescence microscope as well as flow cytometry,described above. Useful dyes to quantify microorganisms include but arenot limited to acridine orange (AO), 4,6-di-amino-2 phenylindole (DAPI)and 5-cyano-2,3 Dytolyl Tetrazolium Chloride (CTC). Viable cells can beestimated by a viability staining method such as the LIVE/DEAD®Bacterial Viability Kit (Bac-Light™) which contains two nucleic acidstains: the green-fluorescent SYTO 9™ dye penetrates all membranes andthe red-fluorescent propidium iodide (PI) dye penetrates cells withdamaged membranes. Therefore, cells with compromised membranes willstain red, whereas cells with undamaged membranes will stain green.Fluorescent in situ hybridization (FISH) extends epifluorescencemicroscopy, allowing for the fast detection and enumeration of specificorganisms. FISH uses fluorescent labelled oligonucleotides probes(usually 15-25 basepairs) which bind specifically to organism DNA in thesample, allowing the visualization of the cells using an epifluorescenceor confocal laser scanning microscope (CLSM). Catalyzed reporterdeposition fluorescence in situ hybridization (CARD-FISH) improves uponthe FISH method by using oligonucleotide probes labelled with a horseradish peroxidase (HRP) to amplify the intensity of the signal obtainedfrom the microorganisms being studied. FISH can be combined with othertechniques to characterize microorganism communities. One combinedtechnique is high affinity peptide nucleic acid (PNA)-FISH, where theprobe has an enhanced capability to penetrate through the ExtracellularPolymeric Substance (EPS) matrix. Another example is LIVE/DEAD-FISHwhich combines the cell viability kit with FISH and has been used toassess the efficiency of disinfection in drinking water distributionsystems.

In another embodiment, the sample, or a portion thereof is subjected toRaman micro-spectroscopy in order to determine the presence of amicroorganism type and the absolute number of at least one microorganismtype (FIG. 1, 1001-1002; FIG. 2, 2001-2002). Raman micro-spectroscopy isa non-destructive and label-free technology capable of detecting andmeasuring a single cell Raman spectrum (SCRS). A typical SCRS providesan intrinsic biochemical “fingerprint” of a single cell. A SCRS containsrich information of the biomolecules within it, including nucleic acids,proteins, carbohydrates and lipids, which enables characterization ofdifferent cell species, physiological changes and cell phenotypes. Ramanmicroscopy examines the scattering of laser light by the chemical bondsof different cell biomarkers. A SCRS is a sum of the spectra of all thebiomolecules in one single cell, indicating a cell's phenotypic profile.Cellular phenotypes, as a consequence of gene expression, usuallyreflect genotypes. Thus, under identical growth conditions, differentmicroorganism types give distinct SCRS corresponding to differences intheir genotypes and can thus be identified by their Raman spectra.

In yet another embodiment, the sample, or a portion thereof is subjectedto centrifugation in order to determine the presence of a microorganismtype and the number of at least one microorganism type (FIG. 1,1001-1002; FIG. 2, 2001-2002). This process sediments a heterogeneousmixture by using the centrifugal force created by a centrifuge. Moredense components of the mixture migrate away from the axis of thecentrifuge, while less dense components of the mixture migrate towardsthe axis. Centrifugation can allow fractionation of samples intocytoplasmic, membrane and extracellular portions. It can also be used todetermine localization information for biological molecules of interest.Additionally, centrifugation can be used to fractionate total microbialcommunity DNA. Different prokaryotic groups differ in theirguanine-plus-cytosine (G+C) content of DNA, so density-gradientcentrifugation based on G+C content is a method to differentiateorganism types and the number of cells associated with each type. Thetechnique generates a fractionated profile of the entire community DNAand indicates abundance of DNA as a function of G+C content. The totalcommunity DNA is physically separated into highly purified fractions,each representing a different G+C content that can be analyzed byadditional molecular techniques such as denaturing gradient gelelectrophoresis (DGGE)/amplified ribosomal DNA restriction analysis(ARDRA) (see discussion herein) to assess total microbial communitydiversity and the presence/quantity of one or more microorganism types.

In another embodiment, the sample, or a portion thereof is subjected tostaining in order to determine the presence of a microorganism type andthe number of at least one microorganism type (FIG. 1, 1001-1002; FIG.2, 2001-2002). Stains and dyes can be used to visualize biologicaltissues, cells or organelles within cells. Staining can be used inconjunction with microscopy, flow cytometry or gel electrophoresis tovisualize or mark cells or biological molecules that are unique todifferent microorganism types. In vivo staining is the process of dyeingliving tissues, whereas in vitro staining involves dyeing cells orstructures that have been removed from their biological context.Examples of specific staining techniques for use with the methodsdescribed herein include, but are not limited to: gram staining todetermine gram status of bacteria, endospore staining to identify thepresence of endospores, Ziehl-Neelsen staining, haematoxylin and eosinstaining to examine thin sections of tissue, papanicolaou staining toexamine cell samples from various bodily secretions, periodicacid-Schiff staining of carbohydrates, Masson's trichome employing athree-color staining protocol to distinguish cells from the surroundingconnective tissue, Romanowsky stains (or common variants that includeWright's stain, Jenner's stain, May-Grunwald stain, Leishman stain andGiemsa stain) to examine blood or bone marrow samples, silver stainingto reveal proteins and DNA, Sudan staining for lipids and Conklin'sstaining to detect true endospores. Common biological stains includeacridine orange for cell cycle determination; bismarck brown for acidmucins; carmine for glycogen; carmine alum for nuclei; Coomassie bluefor proteins; Cresyl violet for the acidic components of the neuronalcytoplasm; Crystal violet for cell walls; DAPI for nuclei; eosin forcytoplasmic material, cell membranes, some extracellular structures andred blood cells; ethidium bromide for DNA; acid fuchsine for collagen,smooth muscle or mitochondria; haematoxylin for nuclei; Hoechst stainsfor DNA; iodine for starch; malachite green for bacteria in the Gimenezstaining technique and for spores; methyl green for chromatin; methyleneblue for animal cells; neutral red for Nissl substance; Nile blue fornuclei; Nile red for lipohilic entities; osmium tetroxide for lipids;rhodamine is used in fluorescence microscopy; safranin for nuclei.Stains are also used in transmission electron microscopy to enhancecontrast and include phosphotungstic acid, osmium tetroxide, rutheniumtetroxide, ammonium molybdate, cadmium iodide, carbohydrazide, ferricchloride, hexamine, indium trichloride, lanthanum nitrate, lead acetate,lead citrate, lead(II) nitrate, periodic acid, phosphomolybdic acid,potassium ferricyanide, potassium ferrocyanide, ruthenium red, silvernitrate, silver proteinate, sodium chloroaurate, thallium nitrate,thiosemicarbazide, uranyl acetate, uranyl nitrate, and vanadyl sulfate.

In another embodiment, the sample, or a portion thereof is subjected tomass spectrometry (MS) in order to determine the presence of amicroorganism type and the number of at least one microorganism type(FIG. 1, 1001-1002; FIG. 2, 2001-2002). MS, as discussed below, can alsobe used to detect the presence and expression of one or more uniquemarkers in a sample (FIG. 1, 1003-1004; FIG. 2, 2003-2004). MS is usedfor example, to detect the presence and quantity of protein and/orpeptide markers unique to microorganism types and therefore to providean assessment of the number of the respective microorganism type in thesample. Quantification can be either with stable isotope labelling orlabel-free. De novo sequencing of peptides can also occur directly fromMS/MS spectra or sequence tagging (produce a short tag that can bematched against a database). MS can also reveal post-translationalmodifications of proteins and identify metabolites. MS can be used inconjunction with chromatographic and other separation techniques (suchas gas chromatography, liquid chromatography, capillary electrophoresis,ion mobility) to enhance mass resolution and determination.

In another embodiment, the sample, or a portion thereof is subjected tolipid analysis in order to determine the presence of a microorganismtype and the number of at least one microorganism type (FIG. 1,1001-1002; FIG. 2, 2001-2002). Fatty acids are present in a relativelyconstant proportion of the cell biomass, and signature fatty acids existin microbial cells that can differentiate microorganism types within acommunity. In one embodiment, fatty acids are extracted bysaponification followed by derivatization to give the respective fattyacid methyl esters (FAMEs), which are then analyzed by gaschromatography. The FAME profile in one embodiment is then compared to areference FAME database to identify the fatty acids and theircorresponding microbial signatures by multivariate statistical analyses.

In the aspects of the methods provided herein, the number of uniquefirst makers in the sample, or portion thereof (e.g., sample aliquot) ismeasured, as well as the abundance of each of the unique first markers(FIG. 1, 1003; FIG. 2, 2003). A unique marker is a marker of amicroorganism strain. It should be understood by one of ordinary skillin the art that depending on the unique marker being probed for andmeasured, the entire sample need not be analyzed. For example, if theunique marker is unique to bacterial strains, then the fungal portion ofthe sample need not be analyzed. As described above, in someembodiments, measuring the absolute abundance of one or more organismtypes in a sample comprises separating the sample by organism type,e.g., via flow cytometry.

Any marker that is unique to an organism strain can be employed herein.For example, markers can include, but are not limited to, small subunitribosomal RNA genes (16S/18S rDNA), large subunit ribosomal RNA genes(23S/25S/28S rDNA), intercalary 5.8S gene, cytochrome c oxidase,beta-tubulin, elongation factor, RNA polymerase and internal transcribedspacer (ITS).

Ribosomal RNA genes (rDNA), especially the small subunit ribosomal RNAgenes, i.e., 18S rRNA genes (18S rDNA) in the case of eukaryotes and 16SrRNA (16S rDNA) in the case of prokaryotes, have been the predominanttarget for the assessment of organism types and strains in a microbialcommunity. However, the large subunit ribosomal RNA genes, 28S rDNAs,have been also targeted. rDNAs are suitable for taxonomic identificationbecause: (i) they are ubiquitous in all known organisms; (ii) theypossess both conserved and variable regions; (iii) there is anexponentially expanding database of their sequences available forcomparison. In community analysis of samples, the conserved regionsserve as annealing sites for the corresponding universal PCR and/orsequencing primers, whereas the variable regions can be used forphylogenetic differentiation. In addition, the high copy number of rDNAin the cells facilitates detection from environmental samples.

The internal transcribed spacer (ITS), located between the 18S rDNA and28S rDNA, has also been targeted. The ITS is transcribed but splicedaway before assembly of the ribosomes The ITS region is composed of twohighly variable spacers, ITS1 and ITS2, and the intercalary 5.8S gene.This rDNA operon occurs in multiple copies in genomes. Because the ITSregion does not code for ribosome components, it is highly variable.

In one embodiment, the unique RNA marker can be an mRNA marker, an siRNAmarker or a ribosomal RNA marker.

Protein-coding functional genes can also be used herein as a uniquefirst marker. Such markers include but are not limited to: therecombinase A gene family (bacterial RecA, archaea RadA and RadB,eukaryotic Rad51 and Rad57, phage UvsX); RNA polymerase β subunit (RpoB)gene, which is responsible for transcription initiation and elongation;chaperonins. Candidate marker genes have also been identified forbacteria plus archaea: ribosomal protein S2 (rpsB), ribosomal proteinS10 (rpsJ), ribosomal protein L1 (rplA), translation elongation factorEF-2, translation initiation factor IF-2, metalloendopeptidase,ribosomal protein L22, ffh signal recognition particle protein,ribosomal protein L4/Lle (rplD), ribosomal protein L2 (rplB), ribosomalprotein S9 (rpsl), ribosomal protein L3 (rplC), phenylalanyl-tRNAsynthetase beta subunit, ribosomal protein L14b/L23e (rplN), ribosomalprotein S5, ribosomal protein S19 (rpsS), ribosomal protein S7,ribosomal protein L16/L10E (rplP), ribosomal protein S13 (rpsM),phenylalanyl-tRNA synthetase α subunit, ribosomal protein L15, ribosomalprotein L25/L23, ribosomal protein L6 (rplF), ribosomal protein L11(rplK), ribosomal protein L5 (rplE), ribosomal protein S12/S23,ribosomal protein L29, ribosomal protein S3 (rpsC), ribosomal proteinS11 (rpsK), ribosomal protein L10, ribosomal protein S8, tRNApseudouridine synthase B, ribosomal protein L18P/L5E, ribosomal proteinS15P/S13e, Porphobilinogen deaminase, ribosomal protein S17, ribosomalprotein L13 (rplM), phosphoribosylformylglycinamidine cyclo-ligase(rpsE), ribonuclease HII and ribosomal protein L24. Other candidatemarker genes for bacteria include: transcription elongation protein NusA(nusA), rpoB DNA-directed RNA polymerase subunit beta (rpoB),GTP-binding protein EngA, rpoC DNA-directed RNA polymerase subunitbeta′, priA primosome assembly protein, transcription-repair couplingfactor, CTP synthase (pyrG), secY preprotein translocase subunit SecY,GTP-binding protein Obg/CgtA, DNA polymerase I, rpsF 30S ribosomalprotein S6, poA DNA-directed RNA polymerase subunit alpha, peptide chainrelease factor 1, rpll 505 ribosomal protein L9, polyribonucleotidenucleotidyltransferase, tsf elongation factor Ts (tsf), rplQ 505ribosomal protein L17, tRNA (guanine-N(1)-)-methyltransferase (rplS),rplY probable 505 ribosomal protein L25, DNA repair protein RadA,glucose-inhibited division protein A, ribosome-binding factor A, DNAmismatch repair protein MutL, smpB SsrA-binding protein (smpB),N-acetylglucosaminyl transferase, S-adenosyl-methyltransferase MraW,UDP-N-acetylmuramoylalanine-D-glutamate ligase, rplS 50S ribosomalprotein L19, rplT 50S ribosomal protein L20 (rplT), ruvA Hollidayjunction DNA helicase, ruvB Holliday junction DNA helicase B, serSseryl-tRNA synthetase, rplU 50S ribosomal protein L21, rpsR 30Sribosomal protein S18, DNA mismatch repair protein MutS, rpsT 30Sribosomal protein S20, DNA repair protein RecN, frr ribosome recyclingfactor (frr), recombination protein RecR, protein of unknown functionUPF0054, miaA tRNA isopentenyltransferase, GTP-binding protein YchF,chromosomal replication initiator protein DnaA, dephospho-CoA kinase,16S rRNA processing protein RimM, ATP-cone domain protein,1-deoxy-D-xylulose 5-phosphate reductoisomerase, 2C-methyl-D-erythritol2,4-cyclodiphosphate synthase, fatty acid/phospholipid synthesis proteinPlsX, tRNA(Ile)-lysidine synthetase, dnaG DNA primase (dnaG), ruvCHolliday junction resolvase, rpsP 30S ribosomal protein S16, RecombinaseA recA, riboflavin biosynthesis protein RibF, glycyl-tRNA synthetasebeta subunit, trmU tRNA(5-methylaminomethyl-2-thiouridylate)-methyltransferase, rpml 50Sribosomal protein L35, hemE uroporphyrinogen decarboxylase, Rodshape-determining protein, rpmA 50S ribosomal protein L27 (rpmA),peptidyl-tRNA hydrolase, translation initiation factor IF-3 (infC),UDP-N-acetylmuramyl-tripeptide synthetase, rpmF 50S ribosomal proteinL32, rplL 50S ribosomal protein L7/L12 (rplL), leuS leucyl-tRNAsynthetase, ligA NAD-dependent DNA ligase, cell division protein FtsA,GTP-binding protein TypA, ATP-dependent Clp protease, ATP-bindingsubunit ClpX, DNA replication and repair protein RecF andUDP-N-acetylenolpyruvoylglucosamine reductase.

Phospholipid fatty acids (PLFAs) may also be used as unique firstmarkers according to the methods described herein. Because PLFAs arerapidly synthesized during microbial growth, are not found in storagemolecules and degrade rapidly during cell death, it provides an accuratecensus of the current living community. All cells contain fatty acids(FAs) that can be extracted and esterified to form fatty acid methylesters (FAMEs). When the FAMEs are analyzed using gaschromatography—mass spectrometry, the resulting profile constitutes a‘fingerprint’ of the microorganisms in the sample. The chemicalcompositions of membranes for organisms in the domains Bacteria andEukarya are comprised of fatty acids linked to the glycerol by anester-type bond (phospholipid fatty acids (PLFAs)). In contrast, themembrane lipids of Archaea are composed of long and branchedhydrocarbons that are joined to glycerol by an ether-type bond(phospholipid ether lipids (PLELs)). This is one of the most widely usednon-genetic criteria to distinguish the three domains. In this context,the phospholipids derived from microbial cell membranes, characterizedby different acyl chains, are excellent signature molecules, becausesuch lipid structural diversity can be linked to specific microbialtaxa.

As provided herein, in order to determine whether an organism strain isactive, the level of expression of one or more unique second markers,which can be the same or different as the first marker, is measured(FIG. 1, 1004; FIG. 2, 2004). Unique first unique markers are describedabove. The unique second marker is a marker of microorganism activity.For example, in one embodiment, the mRNA or protein expression of any ofthe first markers described above is considered a unique second markerfor the purposes of this invention.

In one embodiment, if the level of expression of the second marker isabove a threshold level (e.g., a control level) or at a threshold level,the microorganism is considered to be active (FIG. 1, 1005; FIG. 2,2005). Activity is determined in one embodiment, if the level ofexpression of the second marker is altered by at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, or at least about 30%, as compared to a threshold level, which insome embodiments, is a control level.

Second unique markers are measured, in one embodiment, at the protein,RNA or metabolite level. A unique second marker is the same or differentas the first unique marker.

As provided above, a number of unique first markers and unique secondmarkers can be detected according to the methods described herein.Moreover, the detection and quantification of a unique first marker iscarried out according to methods known to those of ordinary skill in theart (FIG. 1, 1003-1004, FIG. 2, 2003-2004).

Nucleic acid sequencing (e.g., gDNA, cDNA, rRNA, mRNA) in one embodimentis used to determine absolute abundance of a unique first marker and/orunique second marker. Sequencing platforms include, but are not limitedto, Sanger sequencing and high-throughput sequencing methods availablefrom Roche/454 Life Sciences, Illumina/Solexa, Pacific Biosciences, IonTorrent and Nanopore. The sequencing can be amplicon sequencing ofparticular DNA or RNA sequences or whole metagenome/transcriptomeshotgun sequencing.

Traditional Sanger sequencing (Sanger et al. (1977) DNA sequencing withchain-terminating inhibitors. Proc Natl. Acad. Sci. USA, 74, pp.5463-5467, incorporated by reference herein in its entirety) relies onthe selective incorporation of chain-terminating dideoxynucleotides byDNA polymerase during in vitro DNA replication and is amenable for usewith the methods described herein.

In another embodiment, the sample, or a portion thereof is subjected toextraction of nucleic acids, amplification of DNA of interest (such asthe rRNA gene) with suitable primers and the construction of clonelibraries using sequencing vectors. Selected clones are then sequencedby Sanger sequencing and the nucleotide sequence of the DNA of interestis retrieved, allowing calculation of the number of unique microorganismstrains in a sample.

454 pyrosequencing from Roche/454 Life Sciences yields long reads andcan be harnessed in the methods described herein (Margulies et al.(2005) Nature, 437, pp. 376-380; U.S. Pat. Nos. 6,274,320; 6,258,568;6,210,891, each of which is herein incorporated in its entirety for allpurposes). Nucleic acid to be sequenced (e.g., amplicons or nebulizedgenomic/metagenomic DNA) have specific adapters affixed on either end byPCR or by ligation. The DNA with adapters is fixed to tiny beads(ideally, one bead will have one DNA fragment) that are suspended in awater-in-oil emulsion. An emulsion PCR step is then performed to makemultiple copies of each DNA fragment, resulting in a set of beads inwhich each bead contains many cloned copies of the same DNA fragment.Each bead is then placed into a well of a fiber-optic chip that alsocontains enzymes necessary for the sequencing-by-synthesis reactions.The addition of bases (such as A, C, G, or T) trigger pyrophosphaterelease, which produces flashes of light that are recorded to infer thesequence of the DNA fragments in each well. About 1 million reads perrun with reads up to 1,000 bases in length can be achieved. Paired-endsequencing can be done, which produces pairs of reads, each of whichbegins at one end of a given DNA fragment. A molecular barcode can becreated and placed between the adapter sequence and the sequence ofinterest in multiplex reactions, allowing each sequence to be assignedto a sample bioinformatically.

Illumina/Solexa sequencing produces average read lengths of about 25basepairs (bp) to about 300 bp (Bennett et al. (2005) Pharmacogenomics,6:373-382; Lange et al. (2014). BMC Genomics 15, p. 63; Fadrosh et al.(2014) Microbiome 2, p. 6; Caporaso et al. (2012) ISME J, 6, p.1621-1624; Bentley et al. (2008) Accurate whole human genome sequencingusing reversible terminator chemistry. Nature, 456:53-59). Thissequencing technology is also sequencing-by-synthesis but employsreversible dye terminators and a flow cell with a field of oligosattached. DNA fragments to be sequenced have specific adapters on eitherend and are washed over a flow cell filled with specificoligonucleotides that hybridize to the ends of the fragments. Eachfragment is then replicated to make a cluster of identical fragments.Reversible dye-terminator nucleotides are then washed over the flow celland given time to attach. The excess nucleotides are washed away, theflow cell is imaged, and the reversible terminators can be removed sothat the process can repeat and nucleotides can continue to be added insubsequent cycles. Paired-end reads that are 300 bases in length eachcan be achieved. An Illumina platform can produce 4 billion fragments ina paired-end fashion with 125 bases for each read in a single run.Barcodes can also be used for sample multiplexing, but indexing primersare used.

The SOLiD (Sequencing by Oligonucleotide Ligation and Detection, LifeTechnologies) process is a “sequencing-by-ligation” approach, and can beused with the methods described herein for detecting the presence andabundance of a first marker and/or a second marker (FIG. 1, 1003-1004;FIG. 2, 2003-2004) (Peckham et al. SOLiD™ Sequencing and 2-BaseEncoding. San Diego, Calif.: American Society of Human Genetics, 2007;Mitra et al. (2013) Analysis of the intestinal microbiota using SOLiD16S rRNA gene sequencing and SOLiD shotgun sequencing. BMC Genomics,14(Suppl 5): S16; Mardis (2008) Next-generation DNA sequencing methods.Annu Rev Genomics Hum Genet, 9:387-402; each incorporated by referenceherein in its entirety). A library of DNA fragments is prepared from thesample to be sequenced, and are used to prepare clonal bead populations,where only one species of fragment will be present on the surface ofeach magnetic bead. The fragments attached to the magnetic beads willhave a universal P1 adapter sequence so that the starting sequence ofevery fragment is both known and identical. Primers hybridize to the P1adapter sequence within the library template. A set of fourfluorescently labelled di-base probes compete for ligation to thesequencing primer. Specificity of the di-base probe is achieved byinterrogating every 1st and 2nd base in each ligation reaction. Multiplecycles of ligation, detection and cleavage are performed with the numberof cycles determining the eventual read length. The SOLiD platform canproduce up to 3 billion reads per run with reads that are 75 bases long.Paired-end sequencing is available and can be used herein, but with thesecond read in the pair being only 35 bases long. Multiplexing ofsamples is possible through a system akin to the one used by Illumina,with a separate indexing run.

The Ion Torrent system, like 454 sequencing, is amenable for use withthe methods described herein for detecting the presence and abundance ofa first marker and/or a second marker (FIG. 1, 1003-1004; FIG. 2,2003-2004). It uses a plate of microwells containing beads to which DNAfragments are attached. It differs from all of the other systems,however, in the manner in which base incorporation is detected. When abase is added to a growing DNA strand, a proton is released, whichslightly alters the surrounding pH. Microdetectors sensitive to pH areassociated with the wells on the plate, and they record when thesechanges occur. The different bases (A, C, G, T) are washed sequentiallythrough the wells, allowing the sequence from each well to be inferred.The Ion Proton platform can produce up to 50 million reads per run thathave read lengths of 200 bases. The Personal Genome Machine platform haslonger reads at 400 bases. Bidirectional sequencing is available.Multiplexing is possible through the standard in-line molecular barcodesequencing.

Pacific Biosciences (PacBio) SMRT sequencing uses a single-molecule,real-time sequencing approach and in one embodiment, is used with themethods described herein for detecting the presence and abundance of afirst marker and/or a second marker (FIG. 1, 1003-1004; FIG. 2,2003-2004). The PacBio sequencing system involves no amplification step,setting it apart from the other major next-generation sequencingsystems. In one embodiment, the sequencing is performed on a chipcontaining many zero-mode waveguide (ZMW) detectors. DNA polymerases areattached to the ZMW detectors and phospholinked dye-labeled nucleotideincorporation is imaged in real time as DNA strands are synthesized. ThePacBio system yields very long read lengths (averaging around 4,600bases) and a very high number of reads per run (about 47,000). Thetypical “paired-end” approach is not used with PacBio, since reads aretypically long enough that fragments, through CCS, can be coveredmultiple times without having to sequence from each end independently.Multiplexing with PacBio does not involve an independent read, butrather follows the standard “in-line” barcoding model.

In one embodiment, where the first unique marker is the ITS genomicregion, automated ribosomal intergenic spacer analysis (ARISA) is usedin one embodiment to determine the number and identity of microorganismstrains in a sample (FIG. 1, 1003, FIG. 2, 2003) (Ranjard et al. (2003).Environmental Microbiology 5, pp. 1111-1120, incorporated by referencein its entirety for all purposes). The ITS region has significantheterogeneity in both length and nucleotide sequence. The use of afluorescence-labeled forward primer and an automatic DNA sequencerpermits high resolution of separation and high throughput. The inclusionof an internal standard in each sample provides accuracy in sizinggeneral fragments.

In another embodiment, fragment length polymorphism (RFLP) ofPCR-amplified rDNA fragments, otherwise known as amplified ribosomal DNArestriction analysis (ARDRA), is used to characterize unique firstmarkers and the abundance of the same in samples (FIG. 1, 1003, FIG. 2,2003) (Massol-Deya et al. (1995). Mol. Microb. Ecol. Manual. 3.3.2, pp.1-18, incorporated by reference in its entirety for all purposes). rDNAfragments are generated by PCR using general primers, digested withrestriction enzymes, electrophoresed in agarose or acrylamide gels, andstained with ethidium bromide or silver nitrate.

One fingerprinting technique used in detecting the presence andabundance of a unique first marker is single-stranded-conformationpolymorphism (SSCP) (Lee et al. (1996). Appl Environ Microbiol 62, pp.3112-3120; Scheinert et al. (1996). J. Microbiol. Methods 26, pp.103-117; Schwieger and Tebbe (1998). Appl. Environ. Microbiol. 64, pp.4870-4876, each of which is incorporated by reference herein in itsentirety). In this technique, DNA fragments such as PCR productsobtained with primers specific for the 16S rRNA gene, are denatured anddirectly electrophoresed on a non-denaturing gel. Separation is based ondifferences in size and in the folded conformation of single-strandedDNA, which influences the electrophoretic mobility. Reannealing of DNAstrands during electrophoresis can be prevented by a number ofstrategies, including the use of one phosphorylated primer in the PCRfollowed by specific digestion of the phosphorylated strands with lambdaexonuclease and the use of one biotinylated primer to perform magneticseparation of one single strand after denaturation. To assess theidentity of the predominant populations in a given microbial ensemble,in one embodiment, bands are excised and sequenced, or SSCP-patterns canbe hybridized with specific probes. Electrophoretic conditions, such asgel matrix, temperature, and addition of glycerol to the gel, caninfluence the separation.

In addition to sequencing based methods, other methods for quantifyingexpression (e.g., gene, protein expression) of a second marker areamenable for use with the methods provided herein for determining thelevel of expression of one or more second markers (FIG. 1, 1004; FIG. 2,2004). For example, quantitative RT-PCR, microarray analysis, linearamplification techniques such as nucleic acid sequence basedamplification (NASBA) are all amenable for use with the methodsdescribed herein, and can be carried out according to methods known tothose of ordinary skill in the art.

In another embodiment, the sample, or a portion thereof is subjected toa quantitative polymerase chain reaction (PCR) for detecting thepresence and abundance of a first marker and/or a second marker (FIG. 1,1003-1004; FIG. 2, 2003-2004). Specific microorganism strains activityis measured by reverse transcription of transcribed ribosomal and/ormessenger RNA (rRNA and mRNA) into complementary DNA (cDNA), followed byPCR (RT-PCR).

In another embodiment, the sample, or a portion thereof is subjected toPCR-based fingerprinting techniques to detect the presence and abundanceof a first marker and/or a second marker (FIG. 1, 1003-1004; FIG. 2,2003-2004). PCR products can be separated by electrophoresis based onthe nucleotide composition. Sequence variation among the different DNAmolecules influences the melting behaviour, and therefore molecules withdifferent sequences will stop migrating at different positions in thegel. Thus electrophoretic profiles can be defined by the position andthe relative intensity of different bands or peaks and can be translatedto numerical data for calculation of diversity indices. Bands can alsobe excised from the gel and subsequently sequenced to reveal thephylogenetic affiliation of the community members. Electrophoresismethods include, but are not limited to: denaturing gradient gelelectrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE),single-stranded-conformation polymorphism (SSCP), restriction fragmentlength polymorphism analysis (RFLP) or amplified ribosomal DNArestriction analysis (ARDRA), terminal restriction fragment lengthpolymorphism analysis (T-RFLP), automated ribosomal intergenic spaceranalysis (ARISA), randomly amplified polymorphic DNA (RAPD), DNAamplification fingerprinting (DAF) and Bb-PEG electrophoresis.

In another embodiment, the sample, or a portion thereof is subjected toa chip-based platform such as microarray or microfluidics to determinethe abundance of a unique first marker and/or presence/abundance of aunique second marker (FIG. 1, 1003-1004, FIG. 2, 2003-2004). The PCRproducts are amplified from total DNA in the sample and directlyhybridized to known molecular probes affixed to microarrays. After thefluorescently labeled PCR amplicons are hybridized to the probes,positive signals are scored by the use of confocal laser scanningmicroscopy. The microarray technique allows samples to be rapidlyevaluated with replication, which is a significant advantage inmicrobial community analyses. In general, the hybridization signalintensity on microarrays is directly proportional to the abundance ofthe target organism. The universal high-density 16S microarray(PhyloChip) contains about 30,000 probes of 16SrRNA gene targeted toseveral cultured microbial species and “candidate divisions”. Theseprobes target all 121 demarcated prokaryotic orders and allowsimultaneous detection of 8,741 bacterial and archaeal taxa. Anothermicroarray in use for profiling microbial communities is the FunctionalGene Array (FGA). Unlike PhyloChips, FGAs are designed primarily todetect specific metabolic groups of bacteria. Thus, FGA not only revealthe community structure, but they also shed light on the in situcommunity metabolic potential. FGA contain probes from genes with knownbiological functions, so they are useful in linking microbial communitycomposition to ecosystem functions. An FGA termed GeoChipcontains >24,000 probes from all known metabolic genes involved invarious biogeochemical, ecological, and environmental processes such asammonia oxidation, methane oxidation, and nitrogen fixation.

A protein expression assay, in one embodiment, is used with the methodsdescribed herein for determining the level of expression of one or moresecond markers (FIG. 1, 1004; FIG. 2, 2004). For example, in oneembodiment, mass spectrometry or an immunoassay such as an enzyme-linkedimmunosorbant assay (ELISA) is utilized to quantify the level ofexpression of one or more unique second markers, wherein the one or moreunique second markers is a protein.

In one embodiment, the sample, or a portion thereof is subjected toBromodeoxyuridine (BrdU) incorporation to determine the level of asecond unique marker (FIG. 1, 1004; FIG. 2, 2004). BrdU, a syntheticnucleoside analog of thymidine, can be incorporated into newlysynthesized DNA of replicating cells. Antibodies specific for BRdU canthen be used for detection of the base analog. Thus BrdU incorporationidentifies cells that are actively replicating their DNA, a measure ofactivity of a microorganism according to one embodiment of the methodsdescribed herein. BrdU incorporation can be used in combination withFISH to provide the identity and activity of targeted cells.

In one embodiment, the sample, or a portion thereof is subjected tomicroautoradiography (MAR) combined with FISH to determine the level ofa second unique marker (FIG. 1, 1004; FIG. 2, 2004). MAR-FISH is basedon the incorporation of radioactive substrate into cells, detection ofthe active cells using autoradiography and identification of the cellsusing FISH. The detection and identification of active cells atsingle-cell resolution is performed with a microscope. MAR-FISH providesinformation on total cells, probe targeted cells and the percentage ofcells that incorporate a given radiolabelled substance. The methodprovides an assessment of the in situ function of targetedmicroorganisms and is an effective approach to study the in vivophysiology of microorganisms. A technique developed for quantificationof cell-specific substrate uptake in combination with MAR-FISH is knownas quantitative MAR (QMAR).

In one embodiment, the sample, or a portion thereof is subjected tostable isotope Raman spectroscopy combined with FISH (Raman-FISH) todetermine the level of a second unique marker (FIG. 1, 1004; FIG. 2,2004). This technique combines stable isotope probing, Ramanspectroscopy and FISH to link metabolic processes with particularorganisms. The proportion of stable isotope incorporation by cellsaffects the light scatter, resulting in measurable peak shifts forlabelled cellular components, including protein and mRNA components.Raman spectroscopy can be used to identify whether a cell synthesizescompounds including, but not limited to: oil (such as alkanes), lipids(such as triacylglycerols (TAG)), specific proteins (such as hemeproteins, metalloproteins), cytochrome (such as P450, cytochrome c),chlorophyll, chromophores (such as pigments for light harvestingcarotenoids and rhodopsins), organic polymers (such aspolyhydroxyalkanoates (PHA), polyhydroxybutyrate (PHB)), hopanoids,steroids, starch, sulfide, sulfate and secondary metabolites (such asvitamin B12).

In one embodiment, the sample, or a portion thereof is subjected toDNA/RNA stable isotope probing (SIP) to determine the level of a secondunique marker (FIG. 1, 1004; FIG. 2, 2004). SIP enables determination ofthe microbial diversity associated with specific metabolic pathways andhas been generally applied to study microorganisms involved in theutilization of carbon and nitrogen compounds. The substrate of interestis labelled with stable isotopes (such as ¹³C or ¹⁵N) and added to thesample. Only microorganisms able to metabolize the substrate willincorporate it into their cells. Subsequently, ¹³C-DNA and ¹⁵N-DNA canbe isolated by density gradient centrifugation and used for metagenomicanalysis. RNA-based SIP can be a responsive biomarker for use in SIPstudies, since RNA itself is a reflection of cellular activity.

In one embodiment, the sample, or a portion thereof is subjected toisotope array to determine the level of a second unique marker (FIG. 1,1004; FIG. 2, 2004). Isotope arrays allow for functional andphylogenetic screening of active microbial communities in ahigh-throughput fashion. The technique uses a combination of SIP formonitoring the substrate uptake profiles and microarray technology fordetermining the taxonomic identities of active microbial communities.Samples are incubated with a ¹⁴C-labeled substrate, which during thecourse of growth becomes incorporated into microbial biomass. The¹⁴C-labeled rRNA is separated from unlabeled rRNA and then labeled withfluorochromes. Fluorescent labeled rRNA is hybridized to a phylogeneticmicroarray followed by scanning for radioactive and fluorescent signals.The technique thus allows simultaneous study of microbial communitycomposition and specific substrate consumption by metabolically activemicroorganisms of complex microbial communities.

In one embodiment, the sample, or a portion thereof is subjected to ametabolomics assay to determine the level of a second unique marker(FIG. 1, 1004; FIG. 2, 2004). Metabolomics studies the metabolome whichrepresents the collection of all metabolites, the end products ofcellular processes, in a biological cell, tissue, organ or organism.This methodology can be used to monitor the presence of microorganismsand/or microbial mediated processes since it allows associating specificmetabolite profiles with different microorganisms. Profiles ofintracellular and extracellular metabolites associated with microbialactivity can be obtained using techniques such as gaschromatography-mass spectrometry (GC-MS). The complex mixture of ametabolomic sample can be separated by such techniques as gaschromatography, high performance liquid chromatography and capillaryelectrophoresis. Detection of metabolites can be by mass spectrometry,nuclear magnetic resonance (NMR) spectroscopy, ion-mobilityspectrometry, electrochemical detection (coupled to HPLC) and radiolabel(when combined with thin-layer chromatography).

According to the embodiments described herein, the presence andrespective number of one or more active microorganism strains in asample are determined (FIG. 1, 1006; FIG. 2, 2006). For example, strainidentity information obtained from assaying the number and presence offirst markers is analyzed to determine how many occurrences of a uniquefirst marker are present, thereby representing a unique microorganismstrain (e.g., by counting the number of sequence reads in a sequencingassay). This value can be represented in one embodiment as a percentageof total sequence reads of the first maker to give a percentage ofunique microorganism strains of a particular microorganism type. In afurther embodiment, this percentage is multiplied by the number ofmicroorganism types (obtained at step 1002 or 2002, see FIG. 1 and FIG.2) to give the absolute abundance of the one or more microorganismstrains in a sample and a given volume.

The one or more microorganism strains are considered active, asdescribed above, if the level of second unique marker expression at athreshold level, higher than a threshold value, e.g., higher than atleast about 5%, at least about 10%, at least about 20% or at least about30% over a control level.

In another aspect of the invention, a method for determining theabsolute abundance of one or more microorganism strains is determined ina plurality of samples (FIG. 2, see in particular, 2007). For amicroorganism strain to be classified as active, it need only be activein one of the samples. The samples can be taken over multiple timepoints from the same source, or can be from different environmentalsources (e.g., different animals).

The absolute abundance values over samples are used in one embodiment torelate the one or more active microorganism strains, with anenvironmental parameter (FIG. 2, 2008). In one embodiment, theenvironmental parameter is the presence of a second active microorganismstrain. Relating the one or more active microorganism strains to theenvironmental parameter, in one embodiment, is carried out bydetermining the co-occurrence of the strain and parameter by correlationor by network analysis.

In one embodiment, determining the co-occurrence of one or more activemicroorganism strains with an environmental parameter comprises anetwork and/or cluster analysis method to measure connectivity ofstrains or a strain with an environmental parameter within a network,wherein the network is a collection of two or more samples that share acommon or similar environmental parameter. In another embodiment, thenetwork and/or cluster analysis method may be applied to determining theco-occurrence of two or more active microorganism strains in a sample(FIG. 2, 2008). In another embodiment, the network analysis comprisesnonparametric approaches including mutual information to establishconnectivity between variables. In another embodiment, the networkanalysis comprises linkage analysis, modularity analysis, robustnessmeasures, betweenness measures, connectivity measures, transitivitymeasures, centrality measures or a combination thereof (FIG. 2, 2009).In another embodiment, the cluster analysis method comprises building aconnectivity model, subspace model, distribution model, density model,or a centroid model and/or using community detection algorithms such asthe Louvain, Bron-Kerbosch, Girvan-Newman, Clauset-Newman-Moore,Pons-Latapy, and Wakita-Tsurumi algorithms (FIG. 2, 2010).

In one embodiment, the cluster analysis method is a heuristic methodbased on modularity optimization. In a further embodiment, the clusteranalysis method is the Louvain method. See, e.g., the method describedby Blondel et al. (2008). Fast unfolding of communities in largenetworks. Journal of Statistical Mechanics: Theory and Experiment,Volume 2008, October 2008, incorporated by reference herein in itsentirety for all purposes.

In another embodiment, the network analysis comprises predictivemodeling of network through link mining and prediction, collectiveclassification, link-based clustering, relational similarity, or acombination thereof. In another embodiment, the network analysiscomprises differential equation based modeling of populations. Inanother embodiment, the network analysis comprises Lotka-Volterramodeling.

In one embodiment, relating the one or more active microorganism strainsto an environmental parameter (e.g., determining the co-occurrence) inthe sample comprises creating matrices populated with linkages denotingenvironmental parameter and microorganism strain associations.

In one embodiment, the multiple sample data obtained at step 2007 (e.g.,over two or more samples which can be collected at two or more timepoints where each time point corresponds to an individual sample), iscompiled. In a further embodiment, the number of cells of each of theone or more microorganism strains in each sample is stored in anassociation matrix (which can be in some embodiments, an abundancematrix). In one embodiment, the association matrix is used to identifyassociations between active microorganism strains in a specific timepoint sample using rule mining approaches weighted with association(e.g., abundance) data. Filters are applied in one embodiment to removeinsignificant rules.

In one embodiment, the absolute abundance of one or more, or two or moreactive microorganism strains is related to one or more environmentalparameters (FIG. 2, 2008), e.g., via co-occurrence determination.Environmental parameters are chosen by the user depending on thesample(s) to be analyzed and are not restricted by the methods describedherein. The environmental parameter can be a parameter of the sampleitself, e.g., pH, temperature, amount of protein in the sample.Alternatively, the environmental parameter is a parameter that affects achange in the identity of a microbial community (i.e., where the“identity” of a microbial community is characterized by the type ofmicroorganism strains and/or number of particular microorganism strainsin a community), or is affected by a change in the identity of amicrobial community. For example, an environmental parameter in oneembodiment, is the food intake of an animal or the amount of eggsproduced by poultry. In one embodiment, the environmental parameter isthe presence, activity and/or abundance of a second microorganism strainin the microbial community, present in the same sample.

In some embodiments described herein, an environmental parameter isreferred to as a metadata parameter.

Other examples of metadata parameters include but are not limited togenetic information from the host from which the sample was obtained(e.g., DNA mutation information), sample pH, sample temperature,expression of a particular protein or mRNA, nutrient conditions (e.g.,level and/or identity of one or more nutrients) of the surroundingenvironment/ecosystem), susceptibility or resistance to disease, onsetor progression of disease, susceptibility or resistance of the sample totoxins, efficacy of xenobiotic compounds (pharmaceutical drugs),biosynthesis of natural products, or a combination thereof.

For example, according to one embodiment, microorganism strain numberchanges are calculated over multiple samples according to the method ofFIG. 2 (i.e., at 2001-2007). Strain number changes of one or more activestrains over time is compiled (e.g., one or more strains that haveinitially been identified as active according to step 2006), and thedirectionality of change is noted (i.e., negative values denotingdecreases, positive values denoting increases). The number of cells overtime is represented as a network, with microorganism strainsrepresenting nodes and the abundance weighted rules representing edges.Markov chains and random walks are leveraged to determine connectivitybetween nodes and to define clusters. Clusters in one embodiment arefiltered using metadata in order to identify clusters associated withdesirable metadata (FIG. 2, 2008).

In a further embodiment, microorganism strains are ranked according toimportance by integrating cell number changes over time and strainspresent in target clusters, with the highest changes in cell numberranking the highest.

Network and/or cluster analysis method in one embodiment, is used tomeasure connectivity of the one or more strains within a network,wherein the network is a collection of two or more samples that share acommon or similar environmental parameter. In one embodiment, networkanalysis comprises linkage analysis, modularity analysis, robustnessmeasures, betweenness measures, connectivity measures, transitivitymeasures, centrality measures or a combination thereof. In anotherembodiment, network analysis comprises predictive modeling of networkthrough link mining and prediction, social network theory, collectiveclassification, link-based clustering, relational similarity, or acombination thereof. In another embodiment, network analysis comprisesdifferential equation based modeling of populations. In yet anotherembodiment, network analysis comprises Lotka-Volterra modeling.

Cluster analysis method comprises building a connectivity model,subspace model, distribution model, density model, or a centroid model.

Network and cluster based analysis, for example, to carry out methodstep 2008 of FIG. 2, can be carried out via a module. As used herein, amodule can be, for example, any assembly, instructions and/or set ofoperatively-coupled electrical components, and can include, for example,a memory, a processor, electrical traces, optical connectors, software(executing in hardware) and/or the like.

Network Analysis

A network and/or cluster analysis method, in one embodiment, is used tomeasure connectivity of the one or more strains within a network,wherein the network is a collection of two or more samples that share acommon or similar environmental parameter. In one embodiment, networkanalysis comprises linkage analysis, modularity analysis, robustnessmeasures, betweenness measures, connectivity measures, transitivitymeasures, centrality measures or a combination thereof. In anotherembodiment, network analysis comprises predictive modeling of networkthrough link mining and prediction, social network theory, collectiveclassification, link-based clustering, relational similarity, or acombination thereof. In another embodiment, network analysis comprisesmutual information, maximal information coefficient (MIC) calculations,or other nonparametric methods between variables to establishconnectivity. In another embodiment, network analysis comprisesdifferential equation based modeling of populations. In yet anotherembodiment, network analysis comprises Lotka-Volterra modeling.

The environmental parameter can be a parameter of the sample itself,e.g., pH, temperature, amount of protein in the sample. Alternatively,the environmental parameter is a parameter that affects a change in theidentity of a microbial community (i.e., where the “identity” of amicrobial community is characterized by the type of microorganismstrains and/or number of particular microorganism strains in acommunity), or is affected by a change in the identity of a microbialcommunity. For example, an environmental parameter in one embodiment, isthe food intake of an animal or the amount of eggs produced. In oneembodiment, the environmental parameter is the presence, activity and/orabundance of a second microorganism strain in the microbial community,present in the same sample. In some embodiments, an environmentalparameter is referred to as a metadata parameter.

Other examples of metadata parameters include but are not limited togenetic information from the host from which the sample was obtained(e.g., DNA mutation information), sample pH, sample temperature,expression of a particular protein or mRNA, nutrient conditions (e.g.,level and/or identity of one or more nutrients) of the surroundingenvironment/ecosystem), susceptibility or resistance to disease, onsetor progression of disease, susceptibility or resistance of the sample totoxins, efficacy of xenobiotic compounds (pharmaceutical drugs),biosynthesis of natural products, or a combination thereof.

Poultry Pathogen Resistance and Clearance

In some aspects, the present disclosure is drawn to administering one ormore microbial compositions described herein to poultry to clear thegastrointestinal tract of pathogenic microbes. In some embodiments, thepresent disclosure is further drawn to administering microbialcompositions described herein to prevent colonization of pathogenicmicrobes in the gastrointestinal tract. In some embodiments, theadministration of microbial compositions described herein further clearpathogens from the integument and the respiratory tract of fowl, and/orprevent colonization of pathogens on the integument and in therespiratory tract. In some embodiments, the administration of microbialcompositions described herein reduce leaky gut/intestinal permeability,inflammation, and/or incidence of liver disease.

In some embodiments, the microbial compositions of the presentdisclosure comprise one or more microbes that are present in thegastrointestinal tract of poultry at a relative abundance of less than15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, or 0.01%.

In some embodiments, after administration of microbial compositions ofthe present disclosure the one or more microbes are present in thegastrointestinal tract of the poultry at a relative abundance of atleast 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

In some embodiments the administration of microbial compositionsdescribed herein further decrease the prevalence or the likelihood of apathogen to be found in the fowl. In some embodiments, theadministration of microbial compositions described herein furtherdecreases the pathogen abundance in the fowl.

Pathogenic microbes of poultry include the following: Mycoplasmagallisepticum, Mycoplasma meleagridis, Mycoplasma synoviae, Pasteurellamultocida, Clostridium perfringens, Clostridium colinum, Clostridiumbotulinum, Salmonella typi, Salmonella typhimurium, Salmonella enterica,Salmonella pullorum, Salmonella gallinarum, Hemophilus gallinarum,Erysipelothrix insidiosa, Campylobacter jejuni, Campylobacter coli,Campylobacter lari, Listeria monocytogenes, Arcobacter butzleri,Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigellasonnei, Mycobacterium avium, and pathogenic strains of Escherichia coliand Staphylococcus aureus. In some embodiments, the pathogenic microbesinclude viral pathogens. In some embodiments, the pathogenic microbesare pathogenic to both poultry and humans. In some embodiments, thepathogenic microbes are pathogenic to either poultry or humans.

In some embodiments, the administration of compositions of the presentdisclosure to poultry modulate the makeup of the gastrointestinalmicrobiome such that the administered microbes outcompete microbialpathogens present in the gastrointestinal tract. In some embodiments,the administration of compositions of the present disclosure to poultryharboring microbial pathogens outcompetes the pathogens and clears thepoultry of the pathogens. In some embodiments, the administration ofcompositions of the present disclosure stimulate host immunity, and aidsin clearance of the microbial pathogens. In some embodiments, theadministration of compositions of the present disclosure introducemicrobes that produce bacteriostatic and/or bactericidal components thatdecrease or clear the poultry of the microbial pathogens. In someembodiments, the administration of compositions of the presentdisclosure introduces microbes that modulate the pH, nutrientavailability, minteral composition, and/or vitamin composition of thegastrointestinal tract. In some embodiments, the administration ofcompositions of the present disclosure introduces microbes that increasethe gastrointestinal pH, resulting in the inhibition of pathogen growth.In some embodiments, the administration of compositions of the presentdisclosure introduces microbes that decrease the gastrointestinal pH,resulting in the inhibition of pathogen growth.

In some embodiments, challenging poultry with a microbial colonizer ormicrobial pathogen after administering one or more compositions of thepresent disclosure prevents the microbial colonizer or microbialpathogen from growing to a relative abundance of greater than 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or0.01%. In further embodiments, challenging poultry with a microbialcolonizer or microbial pathogen after administering one or morecompositions of the present disclosure prevents the microbial colonizeror microbial pathogen from colonizing poultry

In some embodiments, clearance of the microbial colonizer or microbialpathogen occurs occurs in less than 25 days, less than 24 days, lessthan 23 days, less than 22 days, less than 21 days, less than 20 days,less than 19 days, less than 18 days, less than 17 days, less than 16days, less than 15 days, less than 14 days, less than 13 days, less than12 days, less than 11 days, less than 10 days, less than 9 days, lessthan 8 days, less than 7 days, less than 6 days, less than 5 days, lessthan 4 days, less than 3 days, or less than 2 days post administrationof the one or more compositions of the present disclosure.

In some embodiments, clearance of the microbial colonizer or microbialpathogen occurs within 1-30 days, 1-25 days, 1-20 day, 1-15 days, 1-10days, 1-5 days, 5-30 days, 5-25 days, 5-20 days, 5-15 days, 5-10 days,10-30 days, 10-25 days, 10-20 days, 10-15 days, 15-30 days, 15-25 days,15-20 days, 20-30 days, 20-25 days, or 25-30 days post administration ofthe one or more compositions of the present disclosure.

Improved Traits

In some aspects, the present disclosure is drawn to administeringmicrobial compositions described herein to poultry to improve one ormore traits through the modulation of aspects of weight, musculature,meat characteristics, egg quantity, egg weight, egg volume, egg quality,egg shell density, digestive chemistry, efficiency of feed utilizationand digestibility, fecal output, methane production, overall birdhealth, prevention of colonization of pathogenic microbes, and clearanceof pathogenic microbes.

The lifespane of commercial fowl are somewhat short, 45 days in theinstance of commercial broiler chickens. Short lifespans make thesimilarity of microbial communities of the gastrointestinal tract (GIT)between birds in the same pen low, and between pens very low. The largedifference in microbial communities between the birds over time causesvastly different respondes in feed efficiency (FE), final gain,mortality, and GIT disease outbreaks. The responses are then used asmanagement metrics, which can lead to spurious decisions because of thelarge variance(s) exhibited. By quickly converging the microbialcommunities to a similar final microbial community after hatching, thevariance, in response, is decreased. Furthermore, the quick transitionto a more matured microbeal community contributes to fend off GITpathogen colonization and disease outbreak. This allows for broadresponse observations (i.e., one feed creates higher EE over another) tobe more applicable to broad populations of birds. Ultimately, the lowerthe variance, the greater the uniformity of the flock.

In some embodiments, the increase in egg quantity is an increase of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 eggs relative to an animal nothaving been administered a composition of the present disclosure. Insome embodiments, the increase in egg quantity is an increase of lessthan 2, 3, 4, 5, 6, 7, 8, 9, or 10 eggs relative to an animal not havingbeen administered a composition of the present disclosure. In someembodiments, the increase in egg quantity is an increase of at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%,140%, 150%, 160%, 170%, 180%, 190%, or 200% relative to an animal nothaving been administered a composition of the present disclosure.

In some embodiments, the increase in egg volume is an increase of atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to an animal not havingbeen administered a composition of the present disclosure. In someembodiments, the increase in egg volume is an increase of less than 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% relative to an animal not having beenadministered a composition of the present disclosure.

In some embodiments, the fecal output is reduced by at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to an animal nothaving been administered a composition of the present disclosure. Insome embodiments, the fecal output is reduced by less than 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to an animal nothaving been administered a composition of the present disclosure.

In some embodiments, the fowl having been administered a composition ofthe present disclosure exhibit a weight gain of at least 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a fowl not having beenadministered a composition of the present disclosure.

In some embodiments, the fowl having been administered a composition ofthe present disclosure exhibit a weight gain of at least about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a fowl not havingbeen administered a composition of the present disclosure.

In some embodiments, the fowl having been administered a composition ofthe present disclosure exhibit a feed conversion ratio decrease of atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to afowl not having been administered a composition of the presentdisclosure.

In some embodiments, the fowl having been administered a composition ofthe present disclosure exhibit a feed conversion ratio decrease of atleast about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relativeto a fowl not having been administered a composition of the presentdisclosure.

In some embodiments, the fowl having been administered a composition ofthe present disclosure exhibit a decrease in the number of necroticenteritis-causing bacteria in the gastrointestinal tract of at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a fowl nothaving been administered a composition of the present disclosure.

In some embodiments, the fowl having been administered a composition ofthe present disclosure exhibit a decrease in the number of necroticenteritis-causing bacteria in the gastrointestinal tract of at leastabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to afowl not having been administered a composition of the presentdisclosure.

In some embodiments, the fowl having been administered a composition ofthe present disclosure exhibit a decrease in the number of pathogenicbacteria in the gastrointestinal tract of at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% relative to a fowl not having beenadministered a composition of the present disclosure.

In some embodiments, the fowl having been administered a composition ofthe present disclosure exhibit a decrease in the number of pathogenicbacteria in the gastrointestinal tract of at least about 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a fowl not having beenadministered a composition of the present disclosure.

In some embodiments, improving the eggs produced by poultry isdesirable, wherein the eggs include triglycerides, triacylglycerides,diacylglycerides, monoacylglycerides, phospholipids, cholesterol,glycolipids, and free fatty acids. In further embodiments, free fattyacids include short chain fatty acids (i.e., C4:0, C6:0, and C8:0),medium chain fatty acids (i.e., C10:0, C10:1, C12:0, C14:0, C14:1, andC15:0), and long chain fatty acids (i.e., C16:0, C16:1, C17:0, C17:1,C18:0, C18:1, C18:2, C18:3, and C20:0).

In some embodiments, improving the quantity of vitamins in eggs producedby poultry is desirable. Vitamins found in eggs include B1, B2, B3, B5,B6, B12, choline, biotin, and folic acid.

In some embodiments, improving the quantity of minerals in eggs producedby poultry is desirable. Minerals found in eggs include phosphorous,iodine, selenium, and calcium. Trace amounts of the following may befound in eggs: barium, copper, iron, manganese, nickel, lead, selenium,strontium, vanadium, selenium, rubidium, and zinc.

In some embodiments, increasing or decreasing chicken serum levels ofcalcium, phosphorous, magnesium, triglycerides, cholesterol, andsaccharides is desirable. The modulation of these serum componentsimpact egg traits such as thickness, porosity, density, nutritionalcontent, desirable taste, fat content, cholesterol content, andcoloration.

In some embodiments, improving the efficiency and digestibility ofanimal feed is desirable. In some embodiments, increasing thedegradation of lignocellulosic components from animal feed is desirable.Lignocellulosic components include lignin, cellulose, and hemicellulose.

In some embodiments, increasing the concentration of fatty acids in thegastrointestinal tract is desirable. Fatty acids include acetic acid,propionic acid, and butyric acid. In some embodiments, maintaining thepH balance in the gastrointestinal tract to prevent destruction ofbeneficial microbial ensembles is desirable. In some embodiments,increasing the concentration of lactic acids in the gastrointestinaltract is desirable. Lactic acid is lowers the pH of the surroundingenvironment, including intracellular pH which can disrupt microbialproton motive force. Lactic acid can also permeabilized the outermembrane of gram-negative bacteria such that they exhibit an increasedsusceptibility to antimicrobials.

In some embodiments, decreasing the amount of methane and manureproduced by poultry is desirable

In some embodiments, a decrease in the amount of total manure producedis desirable. In further embodiments, a decrease in the total amount ofphosphorous and/or nitrogen in the total manure produced is desirable.

In some embodiments, improving the feed intake is desirable. In someembodiments, improving the efficiency of nitrogen utilization of thefeed and/or dry matter ingested by poultry is desirable.

In some embodiments, the improved traits of the present disclosure arethe result of the administration of the presently described microbialcompositions. It is thought that the microbial compositions modulate themicrobiome of poultry such that the biochemistry of one or more elementsof the gastrointestinal tract is changed in such a way that thegastrointestinal liquid and solid substratum are more efficiently andmore completely degraded into subcomponents and metabolites than thegastrointestinal tract of poultry not having been administered microbialcompositions of the present disclosure.

In some embodiments, the increase in efficiency and the increase ofdegradation of the gastrointestinal substratum result in an increase inimproved traits of the present disclosure.

In some embodiments, the increase of any one or more of the traits ofthe present disclosure is an increase of about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%,about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 3′7%, about 38%, about 39%,about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about46%, about 4′7%, about 48%, about 49%, about 50%, about 51%, about 52%,about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%,about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%,about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 9′7%, about98%, about 99%, or about 100% relative to the animal not having beenadministered one or more microbial compositions of the presentdisclosure.

In some embodiments, the increase of any one or more of the traits ofthe present disclosure is an increase of at least 0.1%, at least 0.2%,at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, atleast 9%, at least 10%, at least 11%, at least 12%, at least 13%, atleast 14%, at least 15%, at least 16%, at least 17%, at least 18%, atleast 19%, at least 20%, at least 21%, at least 22%, at least 23%, atleast 24%, at least 25%, at least 26%, at least 27%, at least 28%, atleast 29%, at least 30%, at least 31%, at least 32%, at least 33%, atleast 34%, at least 35%, at least 36%, at least 37%, at least 38%, atleast 39%, at least 40%, at least 41%, at least 42%, at least 43%, atleast 44%, at least 45%, at least 46%, at least 47%, at least 48%, atleast 49%, at least 50%, at least 51%, at least 52%, at least 53%, atleast 54%, at least 55%, at least 56%, at least 57%, at least 58%, atleast 59%, at least 60%, at least 61%, at least 62%, at least 63%, atleast 64%, at least 65%, at least 66%, at least 67%, at least 68%, atleast 69%, at least 70%, at least 71%, at least 72%, at least 73%, atleast 74%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 100% relative to the animal not having beenadministered one or more microbial compositions of the presentdisclosure.

In some embodiments, the decrease of any one or more of the traits ofthe present disclosure is a decrease of about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%,about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 3′7%, about 38%, about 39%,about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about46%, about 4′7%, about 48%, about 49%, about 50%, about 51%, about 52%,about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%,about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%,about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 9′7%, about98%, about 99%, or about 100% relative to the animal not having beenadministered one or more microbial compositions of the presentdisclosure.

In some embodiments, the decrease of any one or more of the traits ofthe present disclosure is a decrease of at least 0.1%, at least 0.2%, atleast 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%,at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least9%, at least 10%, at least 11%, at least 12%, at least 13%, at least14%, at least 15%, at least 16%, at least 17%, at least 18%, at least19%, at least 20%, at least 21%, at least 22%, at least 23%, at least24%, at least 25%, at least 26%, at least 27%, at least 28%, at least29%, at least 30%, at least 31%, at least 32%, at least 33%, at least34%, at least 35%, at least 36%, at least 37%, at least 38%, at least39%, at least 40%, at least 41%, at least 42%, at least 43%, at least44%, at least 45%, at least 46%, at least 47%, at least 48%, at least49%, at least 50%, at least 51%, at least 52%, at least 53%, at least54%, at least 55%, at least 56%, at least 57%, at least 58%, at least59%, at least 60%, at least 61%, at least 62%, at least 63%, at least64%, at least 65%, at least 66%, at least 67%, at least 68%, at least69%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or at least 100% relative to the animal not having beenadministered one or more microbial compositions of the presentdisclosure.

Mode of Action: Gastrointestinal Health Improvement and CompetitiveExclusion

The influence of the gastrointestinal microbiome on broiler health iswell known (Roberts, 2015; Yeoman, 2012; Lee (presentation); Oakley,2014)—a healthy intestinal system will improve the overall welfare andperformance of birds in a commercial farm setting. Although the exactroles and mechanisms of individual species within this intricate andcomplicated system are still largely unknown, the overall beneficialeffects of microorganisms on the host's health and performance have beenstudied. The current knowledge of metabolism and mechanisms of actionare summarized below. See FIG. 14. (Pourabedin and Zhao. 2015. FEMSMicrobiol. Lett. 362:fnv122). FIG. 14 depicts a suite of interactionsthat are all modulated by the composition of the gastrointestinal tractwith a well-balanced population of commensal microbes with an adequatesupply of prebiotic compositions. For example, the commensal bacteriaare (1) producing antibacterial compounds to compete with otherorganisms, including pathogens, (2) producing simple fatty acidsinvolved in metabolic regulation and energy use, (3) immunomodulatingthe localized immune responses in conjunction with lymphocytes andantigen presenting cells, etc.

General Nutrition and Gut Health

Increasing the Concentration of Beneficial Molecules, Including ShortChain Fatty Acids and Other Organic Acids, in the Gastrointestinal Tractof the Broiler Improves Bird Performance.

Microbial short chain fatty acid production, in particular, are absorbedand metabolized by the bird and can provide 5% to 15% of the dailyrequirements for bird maintenance energy (Chichlowski, 2007; Annison,1968; Gasaway, 1976ab). Previous studies have shown that supplementationof butyrate can improve both overall weight gain and feed-conversionwhen administered daily to the bird, and that supplementation of anyorganic acid (including fumaric and lactic) can improve bird weight gain(Levy, 2015; Gilliland, 1977; Afil, 2010). Levy, et al. (2015) showedthat improvements in body weight gain and feed conversion increasedlinearly with increasing concentrations of encapsulated butyric acidlevels. Butyrate also enhances vili development (Chamba, 2014) activatesthe immune response, and can also have a direct bactericidal effect(Gantois, 2006).

Improving Development of the Gastrointestinal Tract, Enhancing VilliGrowth, and Stimulating the Immune System.

Supplementation of butyrate and other organic acids to the diets ofbirds have been shown to enhance vili development and stimulate theimmune system (Chamba, 2014; Adil 2010; Adil 2011).

Improving Apparent Metabolizable Energy of the Diet

Fermentation of various microbes can convert carbohydrates to variousend products. Most short chain fatty acids produced by thesemicroorganisms are absorbed and utilized by the bird (Rinttila, 2013;Annison, 1968; Gasaway, 1976ab). The synthesis of vitamins, includingvitamins B and K, are also carried out by microorganisms (Cummings,1997).

Competitive Exclusion

Bacteriocin Production

Microorganisms within the gastrointestinal tract self-regulate throughthe production of various antimicrobial chemicals. Bacteriocins, forexample, are commonly produced by lactic acid microorganisms and canprevent the colonization of pathogens (Chen, 2007; Juven 1990).Short-chain fatty acids been shown to impact and inhibit entericbacteria including Salmonella typhimurium, but do not inhibitbeneficial, native microorganisms (Van der Wielen et al., 2000). Bothpropionic acid, butyric acid, acetate has also been shown to inhibitpathogenic bacteria (Marounek, 1999; Van der Wielen, 2000; Immerseei,2003).

Competitive Use of Nutrients/Binding Sites

Birds are first inoculated with microorganisms shortly after birth. Asthe bird continues to develop, the microbiome colonizes and establishesitself, ultimately creating a stable ecosystems that houses organismsthat occupy all niches and utilizes all available nutrients (Callaway,2008). This expansive, stable community can prevent pathogens fromcolonizing.

Creating environments that are not conducive to pathogen growth

Microorganisms residing within the gut reduce the redox potential withinthe gut, creating an environment suitable for obligate anaerobes toflourish (Cummings, 1997; Chicklowki, 20017; Juven 1990). Lactate andother short chain fatty acid production lowers the pH of thegastrointestinal environment, making it more difficult for pathogens tocolonize and grow (Pourabedin, 2015). Native microorganisms have alsobeen shown to neutralize enterotoxins (M'Sadeq, 2015).

EXAMPLES Example I. Microbial Compositions Associated with Improved FeedEfficiency in Broilers (ASC-15-1 Phase I and II)

The objective of this study was to leverage the Ascus Biosciencestechnology to utilize mutual information to rank the importance ofmicrobial strains residing in the gastrointestinal tract of broilersassociated with improved feed efficiency. For each sample, the presenceand number (cell count) of each microorganism type was determined andintegrated to yield the absolute cell count of each microorganism strainpresent in the samples. The active strains were identified, and allinactive strains were removed from subsequent analysis. The maximalinformation coefficient (MIC) was then determined for all activemicroorganisms as well as relevant performance metadata of each bird.Results were pooled to create a list of all relationships and theircorresponding MIC scores. If the relationship scored below a giventhreshold, the relationship was identified as irrelevant. If therelationship was above a given threshold, the relationship wasidentified as relevant, and is further subjected to network analysis inorder to identify the strains that best influenced desirablephysiological and performance characteristics. In this example, thisapproach was used to identify microorganisms that improve feedefficiency/reduced feed conversion ratio.

Phase 1 comprises the utilization of 216 Cobb 500 broiler chickens over21 study days, with actions/events performed on days 0, 14, 15, 16, 17,18, 19, 20, and 21 (FIG. 3). Phase II comprises the utilization of 216Ross 708 broiler chickens over 21 study days, with actions/eventsperformed on days 0, 14, 15, 16, 17, 18, 19, 20, and 21 (FIG. 4). TheCobb 500 and Ross 708 commercial production broiler chickens were allmale and were ˜1 day of age upon receipt (Day 0); Cobb 500 chickens werefrom Siloam Springs North and Ross 708 chickens were from Siloam SpringsNorth. Chickens were separated into two main groups, 120 were utilizedat day 0 and tagged and placed into floor pens, and 96 were utilized atday 14 and were placed into individual cages.

Phase I and II utilized Test Article I, Coccidiostat (Sacox 60); LotNumber/Expiration: JSB443/August 2017, which is manufactured byHuvepharma Inc. Coccidiostat was commercially available at aconcentration of 60 g/lb with an inclusion level of 50 g/ton, and wasstored in a secured and temperature-monitored dry area. The method ofadministration was via complete feed over a duration of 21 days(Starter). Coccidiostat was administered ad libitum in complete feed.

Phase I utilized Feed Additive I, Phytase 2500 from Nutra Blend, LLC;Lot Number: 06115A07. Phytase 2500 was commercially available at aconcentration of 2,500 FTU/g with an inclusion level of 0.02%, and wasstored in a secured and temperature-monitored dry area. The method ofadministration is via complete feed over a duration of 21 days. Phytase2500 was administered ad libitum in complete feed.

The starter basal diets were manufactured at Colorado Quality Research,Inc. (CQR) feed mill using a standard CQR formulated broiler dietrepresentative of a commercial broiler diet (Industry Standard Average).Basal and treatment diet mixing, pelleting, and crumbling was conductedat CQR using a 500-lb capacity vertical mixer, a 4,000-lb capacityvertical mixer, or a 14,000-lb horizontal mixer and California PelletMill system. Approximately 342 lbs of feed was mixed per treatment. Thefeed was stored in 501b capacity feed sacks and/or bulk storage binslabelled with treatment identity and further identified with a colorcode.

The basal feed and treatment diets were sampled in duplicate (˜300 gsample size). One sample of the basal and each treatment diet wassubmitted to the sponsor for assay and one sample was retained by CQRuntil study end. All samples were labelled with the CQR project number,treatment number, sample description, and date of collection.

Experimental Design Test Groups

Upon placement, chicks were placed into pens based on breed and dietarytreatment. The study was divided into two phases, the aforementionedPhase I and Phase II. The phases took place two weeks apart. The birdswere placed in floor pens by treatment from 0-14D. For each phase, thetest facility was divided into 1 block of 2 pens and 48 blocks of 2individual cages each. Treatments were assigned to the pens/cages usinga complete randomized block design; pens/cages retained their treatmentsthroughout the study. The treatments were identified by numeric codes.Birds were assigned to the cages/pens randomly according to CQR standardoperating procedure B-10. Specific treatment groups were designed asdepicted in Table 12

TABLE 12 Experimental design treatments of Phase I and II, treatment Iand II. No. Floor No. of Treatment No. Birds/ Pens/ Birds/ No. Cages/No. Birds/ Treatment Description Strain Floor Pen Treatment CageTreatment Treatment Phase I 1 0.042% Cobb 60 1 1 48 48 (D 14)Salinomycin 500 60 (D 0) 2 No Cobb 60 1 1 48 48 (D 14) Salinomycin 50060 (D 0) Phase II 1 0.042% Ross 60 1 1 48 48 (D 14) Salinomycin 708 60(D 0) 2 No Ross 60 1 1 48 48 (D 14) Salinomycin 708 60 (D 0)

Housing

Assignment of treatments to cages/pens were conducted using a computerprogram. The computer-generated assignment was as follows: Birds housedin an environmentally control facility in large concrete floor pen (size4′×8′) constructed of solid plastic (4′ tall) with clean litter (SeeFIG. 5). At day 14, 96 birds were moved into cages within the sameenvironmentally controlled facility. Each cage was 24″×18″×24″ (See FIG.6) Lighting was via incandescent lights and a commercial lightingprogram was used. Hours of continuous light for every 24 hour period wasas follows in Table 13.

TABLE 13 Lighting programing for incandescent bird lighting ApproximateHours Approximate Approximate of Continuous Light Light Intensity BirdAge (Days) per 24 Hour Period (Foot Candles) 0-6 23 1.0-1.3  7-21 160.2-0.3

Environmental conditions for the birds (i.e., 0.53 ft² in pen,temperature, lighting, feeder, and water space) was similar for alltreatment groups. In order to prevent bird migration, each pen waschecked to assure no openings greater than 1 inch existed forapproximately 14 inches in height between pens.

Vaccinations

Birds were vaccinated for Mareks at the hatchery. Birds were vaccinatedfor Newcastle and infectious bronchitis by spray application on studyday 0. No other vaccinations, except those in the experimental design,were administered during the study. Records of the vaccinations (vaccinesource, type, lot number, and expiration date) were maintained with thestudy records. No vaccinations or medications other than those disclosedherein were utilized.

Water

Water was provided ad libitum throughout the study. The floor pen waterwas via automatic bell drinkers. The battery cage water was via onenipple waterer. Drinkers were checked twice daily and cleaned as neededto assure a clean water supply to birds at all times.

Feed

Feed was proved ad libitum throughout the study. The floor pen feed wasvia hanging, ˜17-inch diameter tube feeders. The cage feed was via onefeeder trough, 9″×4″. A chick feeder tray was placed in each floor penfor approximately the first 4 days.

Daily Observations

The test facility, pens, and birds were observed at least twice dailyfor general flock condition, lighting, water, feed, ventilation, andunanticipated events. The minimum-maximum temperature of the testfacility was recorded once daily.

Mortality and Culls

Starting on study day 0, any bird that was found dead was removed. Birdsthat were unable to reach feed or water were sacrificed and necropsied.Identification of probable cause of death and necropsy findings wererecorded on the pen mortality record.

Body Weight and Feed Intake

˜96 birds were weighed individually each day (days 14-21). Feedremaining in each cage was weighed and recorded daily from days 14-21.The feed intake for each cage was determined for each day.

Weight Gain and Feed Conversion

Body weight gain on a cage basis and an average body weight gain on atreatment basis was determined from days 14-21. Feed conversion wascalculated for each day and overall for the period of days 14-21 usingthe total feed consumption for the cage divided by bird weight. Averagetreatment feed conversion was determined for the period of days 14-21 byaveraging the individual feed conversions from each cage within thetreatment.

Excreta and Digesta Collection

At days 15, 18, and 21, excreta produced over a 24-hour period wascollected by cage, pooled and dried to measure gross energy values withbomb calorimetry. Gross energy of the feed on day 14 was measured forgross energy to determine apparent metabolic energy (AME). On day 21,each bird was euthanized by cervical dislocation to collect thefollowing using the described procedures (gloves were changed betweeneach bird):

Randomly Select 25% of the Birds:

Make 2 aliquots into 1.5 ml tubes for each location: cecum, smallintestine (anywhere), gizzard, and crop (including mucosal scrapings).One aliquot will contain 150 μl of stop solution (5% phenol & 95%ethanol) to submerge the sample. The second aliquot did not contain stopsolution and was stored at 4° C. for shipping.

Immediately placed the contents of one cecum in a 1.5-ml tube prefilledwith 150 μl stop solution.

Placed the contents of the second cecum into an empty 1.5-ml tube.

Immediately split the contents of the small intestine and placed half in1.5-ml tube prefilled with 150 μl stop solution. Placed the other halfin an empty 1.5-ml tube.

Dissected the gizzard out of the GI tract, removed the contents withforceps, split the contents and placed half in a 1.5-ml tube prefilledwith 150 μl stop solution. Placed the other half in an empty 1.5-mltube.

Dissected the crop out of the GI tract, removed the contents withforceps/scraped out mucosal lining, and placed half in a 1.5-ml tubeprefilled with 150 μl stop solution. Placed the other half in an empty1.5 ml tube.

For the Remaining Birds:

Immediately placed the contents of one cecum in a 1.5-ml tube prefilledwith 150 μl stop solution.

Immediately placed the contents of the small intestine into a 1.5-mltube prefilled with 150 μl stop solution.

Dissected the gizzard out of the GI tract, removed the contents withforceps, and placed in a 1.5-ml tube prefilled with 150 μl stopsolution.

Dissected the crop out of the GI tract, removed the contents withforceps/scrape out mucosal lining, and placed in a 1.5-ml tube prefilledwith 150 μl stop solution.

Store all samples at 4° C. until shipment.

Veterinary Care, Intervention, and Euthanasia

Animals that developed significant concurrent disease, which wereinjured and/or whose condition may have affected the outcome of thestudy were removed from the study and euthanized at the time that thedetermination was made. Six days post challenge, all birds in cages wereremoved and lesion scored.

Scales used in weighing of feed and feed additives were licensed and/orcertified by the State of Colorado. At each use the scales were checkedusing standard weights according to CQR standard operating procedures.

Dispositions Feed

An accounting was maintained of all diets. The amount mixed, used anddiscarded was documented. Unused feed was disposed of either by salvagesale and/or placing into a dumpster for commercial transport to a locallandfill for burial. Disposition was documented in the study records.

Test Animals

An accounting was maintained for birds received for the study. Disposalof mortalities and birds sacrificed during the study and at study endwas discarded to the landfill at study end. Documentation of dispositionwas provided with the study records. No food products derived fromanimals enrolled in this study entered the human food chain.

Data Collected (Phase I)

Average Bird weights (Day 14-21) (Table 14)

Daily Bird Performance Summarized by Treatment (Day 14-21) (FIG. 7)

Data Collected (Phase II)

Average Bird weights (Day 14-21) (Table 15)

Mortality and Removal Weights (Day 14-21) (Table 16)

Daily Bird Performance Summarized by Treatment (Day 14-21) (FIG. 8)

TABLE 14 Phase 1, Cobb 500 performance D 14-21 D 14-21 D 21 D 14 D 14-21Feed Feed Wt Wt Gain Consumed Conver- (kg) (kg) (kg) (kg) sion TreatmentGroup 1 Averages 0.820 0.416 0.404 0.560 1.400 Standard Deviations 0.0780.032 0.061 0.053 0.126 CV's 0.095 0.078 0.150 0.096 0.090 TreatmentGroup 2 Averages 0.831 0.414 0.417 0.554 1.335 Standard Deviations 0.0670.033 0.047 0.046 0.089 CV's 0.081 0.079 0.112 0.082 0.067

TABLE 15 Phase II, Ross 708 performances 708 D 14-21 D 14-21 D 21 D 14 D14-21 Feed Feed Wt Wt Gain Consumed Conver- (kg) (kg) (kg) (kg) sionTreatment Group 1 Averages 0.679 0.378 0.301 0.443 1.503 StandardDeviations 0.058 0.034 0.042 0.063 0.117 CV's 0.085 0.089 0.141 0.1430.078 Treatment Group 2 Averages 0.690 0.384 0.304 0.447 1.515 StandardDeviations 0.085 0.033 0.071 0.075 0.244 CV's 0.123 0.086 0.234 0.1680.161

TABLE 16 Mortality and Removal Weights for Phase II (Day 14-21) Days14-21 No. Total Birds Cause M & R No. Birds Bird Started of MortalityRemoved Wt (kg) Remaining TRT Cage Sex # Day 0 Mortality Removal-1Removal-2 Death Wt (kg) Wt (kg) Days 14-21 Day 14 2 12 M 9109 1 1 0 0BAC- 0.304 0.000 0.304 0 DH 2 20 M 9105 1 1 0 0 BAC- 0.262 0.000 0.262 0DH 1 69 M 4608 1 1 0 0 BAC- 0.360 0.000 0.360 0 NA

GI Sample Preparation and Sequencing:

After collection, the gastrointestinal (GI) samples were centrifuged at4,000 rpm in a swing bucket centrifuge for 20 minutes at 4° C. Thesupernatant was decanted, and an aliquot of each gastrointestinalcontent sample (1-2 mg) was added to a sterile 1.7 mL tube prefilledwith 0.1 mm glass beads. A second aliquot was collected and stored in anempty, sterile 1.7 mL tube for cell counting.

GI samples in empty tubes were stained and put through a flow cytometerto quantify the number of cells of each microorganism type in eachsample. GI samples with glass beads were homogenized with bead beatingto lyse microorganisms. DNA and RNA was extracted and purified from eachsample and prepared for sequencing on an Illumina Miseq. Samples weresequenced using paired-end chemistry, with 300 base pairs sequenced oneach end of the library.

Sequencing Read Processing and Data Analysis:

Sequencing reads were quality trimmed and processed to identifybacterial species present in the GI tract based on a marker gene, 16SrDNA, or ITS1 and/or ITS2. Count data sets and activity datasets wereintegrated with the sequencing reads to determine the absolute cellnumbers of active microbial species within the gastrointestinalmicrobial community. Production characteristics of the broiler overtime, including feed conversion, weight, mortality, and lesion scores,were linked to the distribution of active microorganisms within eachsample over the course of the experiment using mutual information.

Results

One component of the Ascus Biosciences technology utilized in thisapplication leverages mutual information to rank the importance ofnative microbial strains residing in the gastrointestinal tract of theanimal to specific animal traits. The maximal information coefficient(MIC) scores are calculated for all microorganisms and the desiredanimal trait. Relationships were scored on a scale of 0 to 1, with 1representing a strong relationship between the microbial strain and theanimal trait, and 0 representing no relationship. A cut-off based onthis score is used to define useful and non-useful microorganisms withrespect to the improvement of specific traits.

The MICs were calculated between production characteristics, includingindicators for disease such as lesion scores, and the absolute abundanceof each active microorganism. Microorganisms were ranked by MIC score,and microorganisms with the highest MIC scores were selected as the mostrelevant target species. MIC scores of the microbes of the presentdisclosure are recited in Table 1. The greater the MIC score, thegreater the ability of the microbe to confer an improvement in theperformance and GI health of the bird.

Example II. Microbial Compositions of Broilers with Necrotic EnteritisUtilizing a Clostridium perfringens Challenge Model

The objective of this study was to determine the difference in microbialcompositions during necrotic enteritis when challenged with variouslevels of Clostridium perfringens. More specifically, the study soughtto calculate MIC scores for microbes in the gastrointestinal tract ofbroilers challenged with the pathogen. In this instance, the MIC scoreswere calculated between production characteristics, including indicatorsfor disease such as lesion scores and the absolute abundance of eachactive microorganism. Microbes with the highest MIC scores have thegreatest ability to confer an improvement in the gut performance andgastrointestinal health of broilers.

This study utilized 160 Cobb 500 broiler chickens over 21 study days.The Cobb 500 commercial production broiler chickens were all male andwere ˜1 day of age upon receipt (Day 0); Cobb 500 chickens were fromSiloam Springs North. Chickens were separated into four treatments withtwenty birds per pen and two pens per treatment.

The study utilized a feed additive, Phytase 2500 from Nutra Blend, LLC;Lot Number: 06115A07. Phytase 2500 occurred was commercially availableat a concentration of 2,500 FTU/g with an inclusion level of 0.02%, andis stored in a secured and temperature-monitored dry area. The method ofadministration was via feed over a duration of 21 days.

The starter basal diets were manufactured at Colorado Quality Research,Inc. (CQR) feed mill using a standard CQR formulated broiler dietrepresentative of a commercial broiler diet (Industry Standard Average)without medication. Basal and starter diet mixing, pelleting andcrumbling was conducted at CQR using a 500-lb capacity vertical mixer, a4,000-lb capacity vertical mixer, or a 14,000-lb horizontal mixer andCalifornia Pellet Mill system. Approximately 540 lbs of feed was mixedper treatment. The feed was stored in 501b capacity feed sacks and/orbulk storage bins labelled with treatment identity and furtheridentified with a color code.

The basal feed and treatment diets were sampled in duplicate (˜300 gsample size). One sample of the basal and each treatment diet wassubmitted to the sponsor for assay and one sample was retained by CQRuntil study end. All samples were labelled with the CQR project number,treatment number, sample description, and date of collection.

Experimental Design Test Groups

The test facility was divided into 2 blocks of 4 pens. Treatments wereassigned to the pens/cages using a completely randomized block design.Birds were assigned to the pens randomly according to CQR standardoperating procedure B-10. Specific treatment groups were designed asdepicted in Table 18.

TABLE 18 Experimental design for treatments 1-4. NE Chal- No. Treat-lenge Treatment Birds/ No. of No. of Birds/ ment (Y/N) Description PenPens Treatment 1 N Non-Challenged 20 2 40 2 Y Challenged with half 20 240 typical dose (1.25 ml/ bird; 2.0-9.0 × 10⁸ cfu/ml) 3 Y Challengedwith 20 2 40 typical dose (2.5 ml/ bird; 2.0-9.0 × 10⁸ cfu/ml) 4 YChallenged with twice 20 2 40 the typical dose (5 ml/ bird; 2.0-9.0 ×10⁸ cfu/ml) Total 80 8 160

Housing

Assignment of treatments to cages/pens were conducted using a computerprogram. The computer-generated assignment was as follows in Table 19

TABLE 19 Computer selection of treatments to pens. Block Treatment 1Treatment 2 Treatment 3 Treatment 4 B1 4 1 3 2 B2 7 5 8 6

Birds were housed in an environmentally control facility in wooden floorpens (˜4′×4′ minus 2.25 sq. ft for feeder space) providing floor spaceand bird density of ˜0.69 ft²/bird and temperature, lighting, feeder andwater space was similar for all test groups (See FIG. 9). Birds wereplaced in clean pens containing an appropriate depth of wood shavings toprovide a comfortable environment for the chicks. Additional shavingswere added to pens if they became too damp for comfortable conditionsfor the test birds during the study. Lighting was via incandescentlights and a commercial lighting program was used as noted in thefollowing table.

TABLE 20 Lighting programing for incandescent bird lighting (Reproducedfrom Table 11 in previous example) Approximate Hours of ApproximateApproximate Continuous Light Light Intensity Bird Age (Days) per 24 HourPeriod (Foot Candles) 0-6 23 1.0-1.3  7-21 16 0.2-0.3

In order to prevent bird migration and bacterial spread from pen to pen,each pen had a solid (plastic) divider for approximately 24 inches inheight between pens.

Vaccinations

Birds were vaccinated for Mareks at the hatchery. Birds were vaccinatedat CQR for Newcastle and infectious bronchitis by spray application onstudy day 0. No other vaccinations, except those in the experimentaldesign, were administered during the study. Records of the vaccinations(vaccine source, type, lot number, and expiration date) were maintainedwith the study records. No vaccinations or medications other than thosedisclosed herein were utilized.

Water

Water was provided ad libitum throughout the study via one Plassondrinker per pen. Drinkers were checked twice daily and cleaned as neededto assure a clean water supply to birds at all times.

Feed

Feed was proved ad libitum throughout the study via one hanging,˜17-inch diameter tube feeder per pen. A chick feeder tray was placed ineach floor pen for approximately the first 4 days. Birds were placed ontheir respective treatment diets upon receipt (day 0), according to theExperimental Design. Feed added and removed from pens from day 0 tostudy end were weighed and recorded.

Daily Observations

The test facility, pens, and birds were observed at least twice dailyfor general flock condition, lighting, water, feed, ventilation, andunanticipated events. If abnormal conditions or abnormal behavior wasnoted at any of the twice-daily observations they were noted in thestudy records. The minimum-maximum temperature of the test facility wasrecorded once daily.

Pen Cards

There were 2 cards attached to each pen. One card identifies the pennumber and the second will include the treatment number.

Animal Handling

Animals were kept under ideal conditions for livability. The animalswere handled in such a manner as to reduce injuries and unnecessarystress. Humane measures were strictly enforced.

Veterinary Care, Intervention, and Euthanasia

Birds that developed clinically significant concurrent disease unrelatedto the test procedures were, at the discretion of the investigator ordesignee, removed from the study and euthanized in accordance with sitestandard operating procedures. In addition, moribund or injured birdsmay also be euthanized upon authority of a site veterinarian or aqualified technician. Any reasons for withdrawal were documented. In ananimal died, or was removed and euthanized for humane reasons, it wasrecorded on the mortality sheet for the pen and a necropsy performed,and was filed to document the reason for removal. If euthanasia wasdeemed necessary, animals were euthanized via cervical dislocation.

Mortality and Culls

Starting on study day 0, any bird that was found dead was removedweighed and necropsied. Birds that are unable to reach feed or waterwere sacrificed and necropsied. The weight and probable cause of deathand necropsy findings were recorded on the pen mortality record.

Body Weight and Feed Intake

Birds were weighed by pen and individually on approximately days 14 and21. The feed remaining in each pen was weighed and recorded on studydays 14 and 21. The feed intake during days 14-21 were calculated.

Weight Gain and Feed Conversion

Average bird weight, on a pen and individual basis, on each weigh daywas summarized. The average feed conversion was calculated on study day21 using the total feed consumption for the pen divided by the totalweight of surviving birds. Adjusted feed conversion was calculated usingthe total feed consumption in a pen divided by the total weight ofsurviving birds and weight of birds that died or were removed from thatpen.

Digesta Collection

On day 21, each bird was euthanized by cervical dislocation to collectthe following using the described procedures, gloves were changedbetween each bird.

Immediately place the contents of one cecum in a 1.5-ml tube prefilledwith 150 μl stop solution.

Immediately place the contents of the small intestine into a 1.5-ml tubeprefilled with 150 μl stop solution.

Dissect the gizzard out of the GI tract, remove the contents withforceps, and place in a 1.5-ml tube prefilled with 150 μl stop solution.

Dissect the crop out of the GI tract, remove the contents withforceps/scrape out mucosal lining, and place in a 1.5-ml tube prefilledwith 150 μl stop solution.

Store all samples at 4° C. until shipment.

Scales

Scales used in weighing of feed and feed additives were licensed and/orcertified by the State of Colorado. At each use the scales were checkedusing standard weights according to CQR standard operating procedures.

Clostridium perfringens Challenge

Method of Administration

The Clostridium perfringens culture was obtained from MicrobialResearch, Inc. Administration of the C. perfringens (CL-15, Type A, aand (32 toxins) cultures in this study were via the feed. Feed from eachpen's feeder was used to mix with the culture. Prior to placing thecultures in the pens, the treatment feed was removed from the birds forapproximately 4-8 hours. For each pen of birds, a fixed amount based onstudy design of the broth culture at a concentration of approximately2.0-9.0×10⁸ cfu/ml was mixed with a fixed amount of feed (˜25 g/bird) inthe feeder tray and all challenged pens were treated the same. Most ofthe culture-feed was consumed within 1-2 hours. So that birds in alltreatments are treated similar, the groups that are not challenged alsohad the feed removed during the same time period as the challengedgroups. Clostridium Challenge

The C. perfringens culture (CL-15) was grown for ˜5 hours at ˜37° C. influid thioglycollate medium containing starch. CL-15 is a field strainof C. perfringens from a broiler outbreak in Colorado. A fresh brothculture was prepared and used each day. For each pen of birds, a fixedamount of the overnight broth culture was mixed with a fixed amount oftreatment feed in the feeder tray (see administration). The amount offeed, volume, and quantitation of culture inoculum, and number of daysdosed was documented in the final report, and all pens were treated thesame. Birds received the C. perfringens culture for one day (day 17).Quantitation was conducted by Microbial Research, Inc on the culture andresults were documented in the final report. There was no targetmortality for this study.

Lesion Scoring

Four days following the last C. perfringens culture administration, fivebirds were randomly selected from each pen by first bird caught,sacrificed, and intestinal lesions scored for necrotic enteritis.Lesions were scored as follows:

0=normal: No NE lesions, small intestine has normal elasticity (rollsback to normal position after being opened).

1=mild: Small intestinal wall is thin and flaccid (remains flat whenopened and doesn't roll back into normal position after being opened);excess mucus covering mucus membrane.

2=moderate: Noticeable reddening and swelling of the intestinal wall;minor ulceration and necrosis of the intestinal membrane; excess mucus.

3=severe: Extensive area(s) of necrosis and ulceration of the smallintestinal membrane; significant hemorrhage; layer of fibrin andnecrotic debris on the mucus membrane (Turkish towel appearance).

4=dead or moribund: Bird that would likely die within 24 hours and hasNE lesion score of 2 or more.

Dispositions Excess Test Articles

An accounting was maintained of the test articles received and used forthis study. Excess test articles were dispositioned or returned to thesponsor. Documentation was provided with the study records.

Feed

An accounting was maintained of all diets. The amount mixed, used anddiscarded was documented. Unused feed was disposed of either by salvagesale and/or placing into a dumpster for commercial transport to a locallandfill for burial. Disposition was documented in the study records.

Test Animals

An accounting was maintained for birds received for the study. Disposalof mortalities and birds sacrificed during the study and at study endwas discarded to the landfill at study end. Documentation of dispositionwas provided with the study records. No food products derived fromanimals enrolled in this study entered the human food chain.

Data Collected

Mortality and Removal Weights for Cobb 500 Males Spanning Days 0 toStudy End (Table 21).

Average Bird Weights and Performance at Day 14 Summarized by Treatment(Table 22).

Average Bird Weights and Performance at Day 21 Summarized by Treatment(Table 23).

Pen Weights and Feed Conversion for Cobb 500 Males Days 14-21 Summarizedby Treatment (Table 24).

Day 21 NE Lesion Scores for Cobb 500 Males Summarized by Treatment(Table 25).

TABLE 21 Mortality and Removal Weights for Cobb 500 Males Spanning Days0 to Study End Days 0-7 No. Total Birds Cause M & R No. Birds PenStarted of Mortality Added Removed Wt (kg) Remaining Block Trt No. Day 0Mortality Added Removal-1 Removal-2 Death Wt (kg) Wt (kg) Wt (kg) Days0-7 Day 7 1 2 1 20 0.000 20 1 4 2 20 0.000 20 1 3 3 20 0.000 20 1 1 4 200.000 20 2 2 5 20 0.000 20 2 4 6 20 0.000 20 2 1 7 20 0.000 20 2 3 8 200.000 20 Days 7-14 No. Total Birds Cause M & R No. Birds Pen Started ofMortality Removed Wt (kg) Remaining Block Trt No. Day 0 MortalityRemoval-1 Removal-2 Death Wt (kg) Wt (kg) Days 7-14 Day 14 1 2 1 200.000 20 1 4 2 20 0.000 20 1 3 3 20 1 SDS 0.235 0.235 19 1 1 4 20 0.00020 2 2 5 20 0.000 20 2 4 6 20 0.000 20 2 1 7 20 0.000 20 2 3 8 20 0.00020 Days 14-21 Total No. M & R Birds Cause Wt (kg) No. Birds Pen Startedof Mortality Removed Days Remaining Block Trt No. Day 0 MortalityRemoval-1 Removal-2 Death Wt (kg) Wt (kg) 14-21 Day 21 1 2 1 20 3 3NE1.768 1.768 17 1 4 2 20 2 2NE 1.156 1.156 18 1 3 3 20 2 ACT; NE 0.9120.912 17 1 1 4 20 0.000 20 2 2 5 20 2 2NE 1.231 1.231 18 2 4 6 20 3 3NE1.904 1.904 17 2 1 7 20 0.000 20 2 3 8 20 1 NE 0.672 0.672 19

TABLE 22 Average Bird Weights and Performance at Day 14 Summarized byTreatment Day No. No. Day 14 14Avg Adjusted Block Trt Pen Birds BirdsPen Wt Bird Wt Feed Feed No. No. No. Started Mortalities Removed Weighed(kg) (kg) Gain Gain 1 1 1 20 0 0 20 8.731 0.437 1.008 1.008 2 1 1 20 0 020 8.679 0.434 0.993 0.993 Totals & Averages 40 0 0 40 8.705 0.435 1.0011.001 Standard Deviations 0.037 0.002 0.010 0.010 CV's 0.422% 0.422%1.039% 1.039% 1 2 2 20 0 0 20 8.847 0.442 0.986 0.986 2 2 2 20 0 0 208.872 0.444 0.985 0.985 Totals & Averages 40 0 0 40 8.860 0.443 0.9850.985 Standard Deviations 0.018 0.001 0.000 0.000 CV's 0.200% 0.200%0.038% 0.038% 1 3 3 20 1 0 19 8.160 0.429 0.993 0.965 2 3 3 20 0 0 208.866 0.443 0.993 0.993 Totals & Averages 40 1 0 39 8.513 0.436 0.9930.979 Standard Deviations 0.499 0.010 0.000 0.020 CV's 5.864% 2.240%0.006% 2.001% 1 4 4 20 0 0 20 8.423 0.421 0.985 0.985 2 4 4 20 0 0 208.553 0.428 0.996 0.996 Totals & Averages 40 0 0 40 8.488 0.424 0.9910.991 Standard Deviations 0.092 0.005 0.008 0.008 CV's 1.083% 1.083%0.767% 0.767%

TABLE 23 Average Bird Weights and Performance at Day 21 Summarized byTreatment Day 21 No. No. Day 21 Avg Adjusted Block Trt Pen Birds BirdsPen Wt Bird Wt Feed Feed No. No. No. Started Mortalities Removed Weighed(kg) (kg) Gain Gain 1 1 1 20 0 0 20 16.756 0.838 1.004 1.004 2 1 1 20 00 20 16.967 0.848 1.010 1.010 Totals &Averages 40 0 0 40 16.862 0.8431.007 1.007 Standard Deviations 0.149 0.007 0.005 0.005 CV's 0.885%0.885% 0.448% 0.448% 1 2 2 20 3 0 17 14.755 0.868 1.129 1.008 2 2 2 20 20 18 15.102 0.839 1.157 1.070 Totals &Averages 40 5 0 35 14.929 0.8531.143 1.039 Standard Deviations 0.245 0.020 0.020 0.044 CV's 1.644%2.398% 1.754% 4.214% 1 3 3 20 2 0 17 14.129 0.831 1.023 0.961 2 3 3 20 10 19 15.024 0.791 1.126 1.078 Totals &Averages 40 3 0 36 14.577 0.8111.075 1.020 Standard Deviations 0.633 0.029 0.073 0.082 CV's 4.342%3.521% 6.761% 8.086% 1 4 4 20 2 0 18 14.746 0.819 1.066 0.989 2 4 4 20 30 17 13.895 0.817 1.186 1.043 Totals &Averages 40 5 0 35 14.321 0.8181.126 1.016 Standard Deviations 0.602 0.001 0.085 0.039 CV's 4.202%0.162% 7.535% 3.797%

TABLE 24 Pen Weights and Feed Conversion for Cobb 500 Males Days 14-21Summarized by Treatment D 21 D 14-21 Feed Adj. No. No. Avg Avg Conver-Feed Birds Birds D 21 Bird Bird sion Conver- Pen Started Weighed Pen WtWt Gain D 14-21 sion Block Trt No. Day 14 Mortality Added Removal-1Removal-2 D 21 (kg) (kg) (kg) (kg) (kg) 1 1 4 20 0 0 0 0 20 16.756 0.8380.401 0.999 0.999 2 1 7 20 0 0 0 0 20 16.967 0.848 0.414 1.028 1.028Totals & Averages 40 0 0 0 0 40 16.862 0.843 0.408 1.014 1.014 StandardDeviations 0.149 0.007 0.009 0.020 0.020 CV's 0.885% 0.885% 2.280%1.996% 1.996% 1 2 1 20 0 0 0 0 20 14.755 0.738 0.295 1.344 1.034 2 2 520 0 0 0 0 20 15.102 0.755 0.312 1.403 1.171 Totals & Averages 40 0 0 00 40 14.929 0.746 0.303 1.373 1.103 Standard Deviations 0.245 0.0120.011 0.042 0.097 CV's 1.644% 1.644% 3.752% 3.035% 8.786% 1 3 3 19 1 0 00 18 14.129 0.785 0.355 1.066 0.924 2 3 8 20 0 0 0 0 20 15.024 0.7510.308 1.319 1.189 Totals & Averages 39 1 0 0 0 38 14.577 0.768 0.3321.192 1.057 Standard Deviations 0.633 0.024 0.034 0.179 0.187 CV's4.342% 3.107% 10.141% 15.014% 17.707% 1 4 2 20 0 0 0 0 20 14.746 0.7370.316 1.173 0.992 2 4 6 20 0 0 0 0 20 13.895 0.695 0.267 1.490 1.099Totals & Averages 40 0 0 0 0 40 14.321 0.716 0.292 1.332 1.045 StandardDeviations 0.602 0.030 0.035 0.224 0.075 CV's 4.202% 4.202% 11.893%16.809% 7.199%

TABLE 25 Day 21 NE Lesion Scores for Cobb 500 Males Summarized byTreatment Lesion Average Trt Pen No. Bird ID Score Pen Score 1 4 6943 10.6 1 4 6941 1 1 4 6954 1 1 4 6940 0 1 4 6939 0 1 7 2181 0 0.0 1 7 21770 1 7 2176 0 1 7 2173 0 1 7 2186 0 Totals & Averages 10 0.3 0.3 StandardDeviations 0.5 CV's 161.0% 2 1 2127 1 1.4 2 1 2118 1 2 1 2113 1 2 1 21142 2 1 2117 2 2 5 2154 1 2.4 2 5 2171 4 2 5 2167 4 2 5 2162 2 2 5 2156 1Totals & Averages 10 1.9 1.9 Standard Deviations 1.2 CV's 63.0% 3 3 21451 1.40 3 3 2142 3 3 3 2134 1 3 3 2136 1 3 3 2139 1 3 8 6977 1 2.2 3 86980 1 3 8 6989 4 3 8 6978 1 3 8 6994 4 Totals & Averages 10 1.8 1.8Standard Deviations 1.3 CV's 73.1% 4 2 6934 3 2.2 4 2 6928 1 4 2 6920 24 2 6932 3 4 2 6919 2 4 6 6960 3 2.2 4 6 6959 2 4 6 6966 2 4 6 6975 3 46 6956 1 Totals & Averages 10 2.2 2.2 Standard Deviations 0.8 CV's 35.9%

GI Sample Preparation and Sequencing:

After collection, the gastrointestinal (GI) samples were centrifuged at4,000 rpm in a swing bucket centrifuge for 20 minutes at 4° C. Thesupernatant was decanted, and an aliquot of each gastrointestinalcontent sample (1-2 mg) was added to a sterile 1.7 mL tube prefilledwith 0.1 mm glass beads. A second aliquot was collected and stored in anempty, sterile 1.7 mL tube for cell counting.

GI samples in empty tubes were stained and put through a flow cytometerto quantify the number of cells of each microorganism type in eachsample. GI samples with glass beads were homogenized with bead beatingto lyse microorganisms. DNA and RNA was extracted and purified from eachsample and prepared for sequencing on an Illumina Miseq. Samples weresequenced using paired-end chemistry, with 300 base pairs sequenced oneach end of the library.

Sequencing Read Processing and Data Analysis:

Sequencing reads were quality trimmed and processed to identifybacterial species present in the GI tract based on a marker gene, 16SrDNA, or ITS1 and/or ITS2. Count data sets and activity datasets wereintegrated with the sequencing reads to determine the absolute cellnumbers of active microbial species within the gastrointestinalmicrobial community. Production characteristics of the broiler overtime, including feed conversion, weight, mortality, and lesion scores,were linked to the distribution of active microorganisms within eachsample over the course of the experiment using mutual information.

Results

One component of the Ascus Biosciences technology utilized in thisapplication leverages mutual information to rank the importance ofnative microbial strains residing in the gastrointestinal tract of theanimal to specific animal traits. The maximal information coefficient(MIC) scores are calculated for all microorganisms and the desiredanimal trait. Relationships were scored on a scale of 0 to 1, with 1representing a strong relationship between the microbial strain and theanimal trait, and 0 representing no relationship. A cut-off based onthis score is used to define useful and non-useful microorganisms withrespect to the improvement of specific traits.

The MICs were calculated between production characteristics, includingindicators for disease such as lesion scores, and the absolute abundanceof each active microorganism. Microorganisms were ranked by MIC score,and microorganisms with the highest MIC scores were selected as the mostrelevant target species. MIC scores of the microbes of the presentdisclosure are recited in Table 1. The greater the MIC score, thegreater the ability of the microbe to confer an improvement in theperformance and GI health of the bird.

Example III. Media Recipes of the Present Disclosure

Medium Preparation:

Dry reagents for each medium (recipes below) were weighed out, andcombined in a flask. Liquid reagents for each medium, if applicable, arethen added to the flask. DI water was added to the flask to bring themedium to its final volume (typically one liter). The medium wasstirred, and then aliquoted into individual serum bottles or Hungatetubes. Serum bottles were filled with 25 mL or 50 mL of medium, andHungate were filled with 10 mL of medium. The serum bottles/hungate tubewere bubbled with 20:80 CO₂/N₂ for 45 minutes. The bottles were thenstoppered, and autoclaved at 121° C. for 15 minutes. After autoclaving,Cysteine-HCl was added to every bottle to achieve a final concentrationof 1 mM Cysteine-HCl. Any post autoclaving reagents were also added. Allof the post autoclaving reagents were sterile filtered using a 0.22 umfilter prior to addition.

Sample Preparation:

Samples from the gastrointestinal tract of broilers were mixed with 500mL of 1×RAMM and homogenized by vortexing in an anaerobic chamber. Thesamples were then serially diluted and added to the prepared serumbottle/hungate tube. The inoculated bottles were incubated at 37° C. fora minimum of 24 hours. Additional compounds were added to the mediaafter autoclaving when noted: (1) sterile butyric acid was added toachieve a final concentration of 10 mN, (2) glycerol was added toachieve a final concentration of 10 mM, (3) acetic acid was added toachieve a final concentration of 10 mM, (4) a min acid D solution wasadded to achieve a final concentration of 10 mM, and (5) arabinose andxylose solution was added to achieve a final concentration of 10 mM.

For enrichments requiring diluted media, the final media preparation wasdiluted 1:10 with DI water prior to autoclaving. The diluted media wasaliquoted into serum bottles or hungate tubes, and then bubbled under10:80 CO₂/N₂ for 45 minutes to an hour prior to autoclaving.

Media:

TABLE 26 Spirillum Medium Component g/L Bacto Peptone 10 Succinate 1(NH4)2SO4 1 MgSO4 × 7H20 1 10 mM FeCl3 × 6H2O 0.72 mL 100 mM MnSO4 × H2O0.12 mL

TABLE 27 CMC Medium Component g/L Peptone 30 K₂HPO₄ 5 Yeast Extract 5Glucose 4 Cellobiose 1 Maltose 1 Starch 1 L-Cysteine 0.5 Meat Extract 15Rumen Fluid (Clarified) 100 mL Meat Peptone 10 Resazurin (1 g/L)  1 mL

TABLE 28 BL Medium Component g/L Meat Extract 2.4 Protease Peptone 10Bacto peptone 5 Soya Peptone 3 Yeast Extract 5 Liver Extract 3.2Dextrose 10 1M MgSO₄ 7H₂O 1.65 mL 0.05M FeSO₄ 7H₂O 0.7 mL 0.25M NaCl0.675 mL 0.05 mM MnSO₄ 0.7 mL Tween 80 0.53 mL

TABLE 29 Brain Heart Infusion (BHI) Medium Component g/L Brain HeartInfusion 18.5 Dextrose 5

TABLE 30 MRS Medium Component g/L Casein Digest 10.0 Meat Extract 10.0Yeast Extract 5.0 Dextrose 20.0 Tween 80 1.0 mL Sodium Acetate 5.0Ammonium citrate 2.0 1M MgSO₄ 7H₂O 0.8 mL 1M MnSO₄ H₂O 0.296 mL 1MK₂HPO₄ 2.0

TABLE 31 M2GSC Medium Component g/L Beef Extract 10.0 Yeast Extract 2.5NaHCO₃ 4 Cellobiose 2 Starch 2 Glucose 2 (NH₄)₂SO₄ (1M) 5.1 mL MgSO₄7H₂O (0.25M) 0.575 mL K₂HPO₄ (1M) 2 mL KH₂PO₄ (1M) 2.55 mL Clarifiedrumen fluid 100 mL

TABLE 32 Amino Acid D Solution- Place the components in a 100 mL bottleand sterile filer into a 50 mL conical Component g/50 mL Glutamic Acid0.736 g Glycine 0.375 g Proline 0.576 g DI H2O 50 mL

TABLE 33 Arabinose + Xylose Media Addition Component g/100 mL Arabinose1.5013 Xylose 1.5013 DI H2O 100 mL

Example IV. TRIAL 1—In Vivo Evaluation of Ascus Microbial CompositionVs. Control (Both w/ Salinomycin)

Basal and Experimental Diets

The starter, grower and basal diets was manufactured using a feed milland stored in bulk.

Final experimental diet mixing, pelleting, and crumbling was conductedusing a 500-lb capacity vertical mixer, a 4000-lb capacity verticalmixer and/or a 14,000-lb horizontal mixer and a California Pellet Mill.Feed was stored in 50-lb capacity feed sacks and/or bulk storage binslabeled with treatment code. Phytase and Sacox 60 was included in alldiets throughout the experiment.

The feeding schedule utilized two feeds a starter feed in crumble formand a grower feed in pellet form. The starter feed was fed from days 0to 17, and the grower feed was fed from days 17 to 35.

Test System

Species Broiler Chicken Strain Commercial production Breed/Cross Cobb500 Supplier Cobb Vantress, Inc Sex Males Age ~1 day of age upon receipt(day 0) ~35 days at final weights Identification Pen cards Number ofbirds: 800 (D 0) Number of treatments:  2 Number of pens/treatment: 20Number of birds/pen:  20 (D 0) Number of birds/treatment: 400 (D 0)Total number of pens: 40

Test Groups

Treatments were assigned to the pens using a complete randomized blockdesign. Treatments were administered to the pens at start of study day0. The treatments will identified by numeric codes. Specific treatmentgroups are as follows. There were two treatment groups, 1 and 2. Eachgroup consisted of twenty birds per pen with a total of 20 pens. Thetotal number of birds per treatment was 400. Treatment 1 consisted ofnon-challenged birds. Treatment 2 consisted of treatment withAscusbbr_5796, Ascusbbr_38717, and Ascusbbr_331885. The Ascus microbialensembles were administered to the birds in the treatment group viadrinking water daily.

Housing and Management

Treatments were randomly assigned to each pen using Microsoft Excelrandom number generator by the Data Manager. Birds were assigned to thepens randomly.

Birds were housed within an environmentally controlled in concrete floorpens providing floor space & bird density of [˜0.55 ft²/bird (day 0);˜0.69 ft²/bird (day 21 after lesion scores)], temperature, humidity,lighting, feeder and water space were similar for all test groups. Birdswere placed in clean pens containing an appropriate depth of clean woodshavings to provide a comfortable environment for the chicks. Additionalshavings were added to pens in order to maintain bird comfort. Lightingwas via incandescent lights and a commercial lighting program was usedas follows.

TABLE 34 Housing Description Approximate Hours Approximate of ContinuousLight ~Light Intensity Bird Age (days) per 24 hr period (foot candles)0-4 24 1.0-1.3  5-10 10 1.0-1.3 11-18 12 0.2-0.3  19-end 16 0.2-0.3

Environmental conditions for the birds (i.e. bird density, temperature,lighting, feeder and water space) were similar for all treatment groups.In order to prevent bird migration and bacterial spread from pen to pen,each pen will have a solid wood or plastic divider for approximately 24inches in height between pens.

Vaccinations and Therapeutic Medication

Birds were vaccinated for Mareks at the hatchery. Upon receipt (studyday 0), birds were vaccinated for Newcastle and Infectious Bronchitisand Coccivac by spray application using a spray cabinet. Documentationof vaccine manufacturer, lot number and expiration date was providedwith the final report.

Water

Water was provided ad libitum throughout the study via one automaticnipple drinker (4 nipples per drinker) per pen. Drinkers were checkedtwice daily and cleaned as needed to assure a clean and constant watersupply to the birds.

Feed

Feed was provided ad libitum throughout the study via one hanging,˜17-inch diameter tube feeder per pen. A chick feeder tray was placed ineach pen for approximately the first 4 days. Birds were placed on theirrespective treatment diets upon receipt (day 0) according to theExperimental Design. Feed added and removed from pens from day 0 tostudy end was weighed and recorded.

Daily Observations

The test facility, pens and birds were observed at least twice daily forgeneral flock condition, lighting, water, feed, ventilation andunanticipated events. If abnormal conditions or abnormal behavior isnoted at any of the twice-daily observations they were documented andincluded with the study records. The minimum-maximum temperature of thetest facility was recorded once daily.

Pen Cards

There were 2 cards attached to each pen. One card identifies the pennumber and the second will include the treatment number.

Animal Handling

The animals were kept under ideal conditions for livability. The animalswere handled in such a manner as to reduce injuries and unnecessarystress. Humane measures were strictly enforced.

Veterinary Care, Intervention and Euthanasia

Birds that develop clinically significant concurrent disease unrelatedto the test procedures may, at the discretion of the Study Investigator,or a designee, be removed from the study and euthanized in accordancewith site SOPs. In addition, moribund or injured birds may also beeuthanized upon authority of a Site Veterinarian or a qualifiedtechnician. The reason for withdrawal was documented. If an animal dies,or is removed and euthanized for humane reasons, it was recorded on themortality sheet for the pen and a necropsy performed and was filed todocument the reason for removal. If euthanasia is deemed necessaryanimals were euthanized by cervical dislocation.

Mortality and Culls

From Day 0 to study end any bird that is found dead or is sacrificed wasweighed and necropsied. The weight and probable cause of death andnecropsy findings were recorded on the mortality record. If sex-slipsare noted at any time during the study they were removed, weighed,necropsied to confirm sex and recorded on the pen mortality record.

Body Weights and Feed Intake

Birds were weighed by pen on approximately day 0, 17, 28 and 35. Thefeed remaining in each pen was weighed and recorded on study days 17, 28and 35. The feed intake during days 0-17, 17-28, and 0-35 wascalculated.

Weight Gains and Feed Conversion

Average bird weight, on a pen basis, on each weigh day was summarized.Bird weight gain by pen days 17-28 was calculated. The average feedconversion was calculated on the study days 17 and 28 (i.e. days 0-17,17-35, and 0-35) using the total feed consumption for the pen divided bythe total weight of surviving birds. Adjusted feed conversion wascalculated using the total feed consumption in a pen divided by thetotal weight of surviving birds and weight of birds that died or wereremoved from that pen.

TABLE 35 Results Avg Individual Adj. Feed Treatment Trt Group Bird WtGain (kg) Conversion Description 1 2.304 1.420 Non- Challenged withsalinomycin 2 2.399 1.407 Treated with salinomycin and AscusComposition: Ascusbbr_5796, Ascusbbr_38717, Ascusbbr_331885

The birds were treated with a composition of Ascus microorganisms todetermine their effects on performance. Three microorganisms,Ascusbbr_5796, Ascusbbr_38717, and Ascusbbr_331885 were administereddaily to the experimental birds via their drinking water over the courseof the entire experiment. All birds were on a commercially relevantpelleted feed that included salinomycin.

At the end of the experiment, birds were sacrificed and weighed. Feedconversion was calculated based on the total feed consumption for thepen divided by the total weight of the surviving birds. The treatmentgroup was found to have a slight improvement in feed conversion (1%) andindividual bird weight gain (4%) as compared to the control group.

Example V. TRIAL 2—In Vivo Evaluation of Ascus Microbial Composition Vs.Clostridium perfringens Challenge

Basal and Experimental Diets

The starter, grower and basal diets was manufactured using a feed milland stored in bulk.

Final experimental diet mixing, pelleting, and crumbling was conductedusing a 500-lb capacity vertical mixer, a 4000-lb capacity verticalmixer and/or a 14,000-lb horizontal mixer and a California Pellet Mill.Feed was stored in 50-lb capacity feed sacks and/or bulk storage binslabeled with treatment code. Phytase was included in all dietsthroughout the experiment.

The feeding schedule utilized two feeds a starter feed in crumble formand a grower feed in pellet form. The starter feed was fed from days 0to 17, and the grower feed was fed from days 17 to 35. The Ascusmicrobial ensembles were administered to the birds in the treatmentgroup via drinking water daily.

Test System

Species Broiler Chicken Strain Commercial production Breed/Cross Cobb500 Supplier TBD Sex Males Age ~1 day of age upon receipt (day 0) ~35days at final weights Identification Pen cards Number of birds: 900 (D0) Number of treatments:  3 Number of pens/treatment: 12 Number ofbirds/pen:  25 (D 0) Number of birds/treatment: 300 (D 0) Total numberof pens: 36

Test Groups

Treatments were assigned to the pens using a complete randomized blockdesign. Treatments were administered to the pens at start of study day0. The treatments will identified by numeric codes. Challenged controltreatments comprise the administration of pathogens as the control.Challenged Ascus compositions comprise the administration ofexperimental microbes. Specific treatment groups are as follows:

TABLE 36 Test Groups CP Chal- Treatment No. of No. of Number of Trtlenged Description Birds/Pen Pens Birds/Trt 1 Yes Challenged Control 2512 300 (non-medicated) 2 Yes Challenged Control 25 12 300 w/salinomycin3 Yes Challenged, Ascus 25 12 300 Composition administered:Ascusbbr_4729, Ascusbbr_331885, Ascusbbr_170211 (water application)

Housing and Management

Housing

Treatments were randomly assigned to each pen using Microsoft Excelrandom number generator by the Data Manager. Birds were assigned to thepens randomly. Birds were housed within an environmentally controlled inconcrete floor pens providing floor space & bird density of [˜0.55ft²/bird (day 0); ˜0.69 ft²/bird (day 21 after lesion scores)],temperature, humidity, lighting, feeder and water space were similar forall test groups. Birds were placed in clean pens containing anappropriate depth of clean wood shavings to provide a comfortableenvironment for the chicks. Additional shavings were added to pens inorder to maintain bird comfort. Lighting was via incandescent lights anda commercial lighting program was used as follows.

TABLE 37 Lighting Approximate Hours of Approximate Continuous Light~Light Intensity Bird Age (days) per 24 hr period (foot candles) 0-4 241.0-1.3  5-10 10 1.0-1.3 11-18 12 0.2-0.3  19-end 16 0.2-0.3

Environmental conditions for the birds (i.e. bird density, temperature,lighting, feeder and water space) were similar for all treatment groups.In order to prevent bird migration and bacterial spread from pen to pen,each pen will have a solid wood or plastic divider for approximately 24inches in height between pens.

Vaccinations and Therapeutic Medication

Birds were vaccinated for Mareks at the hatchery. Upon receipt (studyday 0), birds were vaccinated for Newcastle and Infectious Bronchitisand Coccivac by spray application using a spray cabinet. Documentationof vaccine manufacturer, lot number and expiration date was providedwith the final report.

Water

Water was provided ad libitum throughout the study via one automaticnipple drinker (4 nipples per drinker) per pen. Drinkers were checkedtwice daily and cleaned as needed to assure a clean and constant watersupply to the birds.

Feed

Feed was provided ad libitum throughout the study via one hanging,˜17-inch diameter tube feeder per pen. A chick feeder tray was placed ineach pen for approximately the first 4 days. Birds were placed on theirrespective treatment diets upon receipt (day 0) according to theExperimental Design. Feed added and removed from pens from day 0 tostudy end was weighed and recorded.

Daily Observations

The test facility, pens and birds were observed at least twice daily forgeneral flock condition, lighting, water, feed, ventilation andunanticipated events. If abnormal conditions or abnormal behavior isnoted at any of the twice-daily observations they were documented andincluded with the study records. The minimum-maximum temperature of thetest facility was recorded once daily.

Pen Cards

There were 2 cards attached to each pen. One card identifies the pennumber and the second will include the treatment number.

Animal Handling

The animals were kept under ideal conditions for livability. The animalswere handled in such a manner as to reduce injuries and unnecessarystress. Humane measures were strictly enforced.

Veterinary Care, Intervention and Euthanasia

Birds that develop clinically significant concurrent disease unrelatedto the test procedures may, at the discretion of the Study Investigator,or a designee, be removed from the study and euthanized in accordancewith site SOPs. In addition, moribund or injured birds may also beeuthanized upon authority of a Site Veterinarian or a qualifiedtechnician. The reason for withdrawal was documented. If an animal dies,or is removed and euthanized for humane reasons, it was recorded on themortality sheet for the pen and a necropsy performed and was filed todocument the reason for removal. If euthanasia is deemed necessaryanimals were euthanized by cervical dislocation.

Mortality and Culls

From Day 0 to study end any bird that is found dead or is sacrificed wasweighed and necropsied. The weight and probable cause of death andnecropsy findings were recorded on the mortality record. If sex-slipsare noted at any time during the study they were removed, weighed,necropsied to confirm sex and recorded on the pen mortality record.

Body Weights and Feed Intake

Birds were weighed by pen on approximately day 0, 17, 28 and 35. Thefeed remaining in each pen was weighed and recorded on study days 17, 28and 35. The feed intake during days 0-17, 17-28, and 0-35 wascalculated.

Weight Gains and Feed Conversion

Average bird weight, on a pen basis, on each weigh day was summarized.Bird weight gain by pen days 17-28 was calculated. The average feedconversion was calculated on the study days 17 and 28 (i.e. days 0-17,17-35, and 0-35) using the total feed consumption for the pen divided bythe total weight of surviving birds. Adjusted feed conversion wascalculated using the total feed consumption in a pen divided by thetotal weight of surviving birds and weight of birds that died or wereremoved from that pen.

Coccidiosis Challenge

All birds each received a 1× dose of Coccivac by spray cabinet onapproximately study day 0.

Clostridium perfringens Challenge

Clostridium Challenge:

The Clostridium perfringens culture (CL-15) was grown ˜5 hrs at ˜37° C.in Fluid

Thioglycollate medium containing starch. CL-15 is a field strain ofClostridium perfringens from a broiler outbreak in Colorado. For eachpen of birds, a fixed amount of the broth culture (˜2-3 ml/bird) wasmixed with a fixed amount of treatment feed (˜25 g/bird) in the feedertray. The amount of feed, volume and quantitation of culture inoculum,and number of days dosed were documented in the final report and allpens were treated the same. Birds will receive the C. perfringensculture for one day (Study day 17). The target is 10% mortality with aminimum 5% in the challenged, non-medicated group.

Method of Administration

Administration of the Clostridium perfringens (CL-15, Type A, a and (32toxins) cultures in this study was via the feed. Feed from each pen'sfeeder was used to mix with the culture. Prior to placing the culturesin the pens the treatment feed was removed from the birds forapproximately 4-8 hours. For each pen of birds, a fixed amount (˜2.5ml/bird) of the broth culture at a concentration of approximately2.0-9.0×10⁸ cfu/ml was mixed with a fixed amount of feed (˜25 g/bird) inthe feeder tray and all challenged pens were treated the same. Most ofthe culture-feed was consumed within 1-2 hours. So that birds in alltreatments are treated similar, the groups that are not challenged willalso have the feed removed during the same time period as the challengedgroups.

Lesion Scoring

On study day 21, 5 birds were randomly selected from each pen (by firstbird caught), sacrificed and evaluated for intestinal lesions scored fornecrotic enteritis. Lesions were scored as follows:

0=normal: no NE lesions, small intestine has normal elasticity (rollsback to normal position after being opened)

1=mild: small intestinal wall was thin and flaccid (remains flat whenopened and doesn't roll back into normal position after being opened);excess mucus covering mucus membrane

2=moderate: noticeable reddening and swelling of the intestinal wall;minor ulceration and necrosis of the intestine membrane; excess mucus

3=severe: extensive area(s) of necrosis and ulceration of the smallintestinal membrane; significant hemorrhage; layer of fibrin andnecrotic debris on the mucus membrane (Turkish towel appearance)

4=dead or moribund: bird that would likely die within 24 hours and hasNE lesion score of 2 or more; or birds that died due to necroticenteritis.

TABLE 38 Results Avg Individual Adj. Feed NE- Lesion Trt Group Bird WtGain (kg) Conversion Mortality Score 1 2.020 1.801 54.6% 3.57 2 2.1471.505 3.3% 0.25 3 1.952 1.613 44.6% 3.88

The birds were treated with a composition of Ascus microorganisms todetermine their effects on performance and the prevention of Clostridiumperfrigens infection. Three microorganisms, Ascusbbr_4729,Ascusbbr_331885, and Ascusbbr_170211 were administered daily to theexperimental birds via their drinking water over the course of theentire experiment. All birds were on a commercially relevant pelletedfeed.

Birds were challenged with C. perfringens on day 17 of the study. On day21, 5 birds were randomly selected, sacrified, and lesion scored.Mortality and feed intake were measured throughout the experiment. Atthe end of the experiment, birds were sacrificed and weighed. Feedconversion was calculated based on the total feed consumption for thepen divided by the total weight of the surviving birds. The treatmentgroup receiving Ascus microorganisms was found to have improved feedconversion (10.4%) and percent mortality (18.3%) when compared to thechallenged control. However, lesion scores (8.6%) were higher in theAscus microorganism group compared to to the challenged control. Thesalinomycin control outperformed all groups.

Example VI. TRIAL 3—In Vivo Evaluation of Ascus Microbial CompositionVs. Clostridium perfringens Challenge

Basal and Experimental Diets

The starter, grower and basal diets was manufactured using a feed milland stored in bulk. Final experimental diet mixing, pelleting, andcrumbling was conducted using a 500-lb capacity vertical mixer, a4000-lb capacity vertical mixer and/or a 14,000-lb horizontal mixer anda California Pellet Mill. Feed was stored in 50-lb capacity feed sacksand/or bulk storage bins labeled with treatment code. Phytase wasincluded in all diets throughout the experiment.

The feeding schedule utilized two feeds a starter feed in crumble formand a grower feed in pellet form. The starter feed was fed from days 0to 17, and the grower feed was fed from days 17 to 35. The Ascusmicrobial ensembles were administered to the birds in the treatmentgroup once prior to placement via spray application.

Test System

Species Broiler Chicken Strain Commercial production Breed/Cross Cobb500 Supplier TBD Sex Males Age ~1 day of age upon receipt (day 0) ~35days at final weights Identification Pen cards Number of birds: 1200 (D0) Number of treatments:  4 Number of pens/treatment: 12 Number ofbirds/pen:  25 (D 0) Number of birds/treatment:  300 (D 0) Total numberof pens: 48

Test Groups

Treatments were assigned to the pens using a complete randomized blockdesign. Treatments were administered to the pens at start of study day0. The treatments will identified by numeric codes. Challenged controltreatments comprise the administration of pathogens as the control.Challenged Ascus compositions comprise the administration ofexperimental microbes. Specific treatment groups are as follows.

TABLE 39 Test Groups CP Chal- Treatment No. of No. of Number of Trtlenged Description Birds/Pen Pens Birds/Trt 1 Yes Challenged Control 258 200 (Non-medicated) 2 Yes Challenged Control 25 8 200 with Salinomycin3 Yes Challenged, Ascus 25 8 200 Composition administered:Ascusbbr_4729, Ascusbbr_331885, Ascusbbr_170211 (spray application) 4Yes Challenged, Ascus 25 8 200 Composition administered: Ascusbbr_4729,Ascusbbr_33, Ascusbbr_127 (spray application)

Housing and Management

Housing

Treatments were randomly assigned to each pen using Microsoft Excelrandom number generator by the Data Manager. Birds were assigned to thepens randomly.

Birds were housed within an environmentally controlled in concrete floorpens providing floor space & bird density of [˜0.55 ft²/bird (day 0);˜0.69 ft²/bird (day 21 after lesion scores)], temperature, humidity,lighting, feeder and water space were similar for all test groups. Birdswere placed in clean pens containing an appropriate depth of clean woodshavings to provide a comfortable environment for the chicks. Additionalshavings were added to pens in order to maintain bird comfort. Lightingwas via incandescent lights and a commercial lighting program was usedas follows.

TABLE 40 Lighting Approximate Hours of Approximate Continuous Light~Light Intensity Bird Age (days) per 24 hr period (foot candles) 0-4 241.0-1.3  5-10 10 1.0-1.3 11-18 12 0.2-0.3  19-end 16 0.2-0.3

Environmental conditions for the birds (i.e. bird density, temperature,lighting, feeder and water space) were similar for all treatment groups.In order to prevent bird migration and bacterial spread from pen to pen,each pen will have a solid wood or plastic divider for approximately 24inches in height between pens.

Vaccinations and Therapeutic Medication

Birds were vaccinated for Mareks at the hatchery. Upon receipt (studyday 0), birds were vaccinated for Newcastle and Infectious Bronchitisand Coccivac by spray application using a spray cabinet. Documentationof vaccine manufacturer, lot number and expiration date was providedwith the final report.

Water

Water was provided ad libitum throughout the study via one automaticnipple drinker (4 nipples per drinker) per pen. Drinkers were checkedtwice daily and cleaned as needed to assure a clean and constant watersupply to the birds.

Feed

Feed was provided ad libitum throughout the study via one hanging,˜17-inch diameter tube feeder per pen. A chick feeder tray was placed ineach pen for approximately the first 4 days. Birds were placed on theirrespective treatment diets upon receipt (day 0) according to theExperimental Design. Feed added and removed from pens from day 0 tostudy end was weighed and recorded.

Daily Observations

The test facility, pens and birds were observed at least twice daily forgeneral flock condition, lighting, water, feed, ventilation andunanticipated events. If abnormal conditions or abnormal behavior isnoted at any of the twice-daily observations they were documented andincluded with the study records. The minimum-maximum temperature of thetest facility was recorded once daily.

Pen Cards

There were 2 cards attached to each pen. One card identifies the pennumber and the second will include the treatment number.

Animal Handling

The animals were kept under ideal conditions for livability. The animalswere handled in such a manner as to reduce injuries and unnecessarystress. Humane measures were strictly enforced.

Veterinary Care, Intervention and Euthanasia

Birds that develop clinically significant concurrent disease unrelatedto the test procedures may, at the discretion of the Study Investigator,or a designee, be removed from the study and euthanized in accordancewith site SOPs. In addition, moribund or injured birds may also beeuthanized upon authority of a Site Veterinarian or a qualifiedtechnician. The reason for withdrawal was documented. If an animal dies,or is removed and euthanized for humane reasons, it was recorded on themortality sheet for the pen and a necropsy performed and was filed todocument the reason for removal. If euthanasia is deemed necessaryanimals were euthanized by cervical dislocation.

Mortality and Culls

From Day 0 to study end any bird that is found dead or is sacrificed wasweighed and necropsied. The weight and probable cause of death andnecropsy findings were recorded on the mortality record. If sex-slipsare noted at any time during the study they were removed, weighed,necropsied to confirm sex and recorded on the pen mortality record.

Body Weights and Feed Intake

Birds were weighed by pen on approximately day 0, 17, 28 and 35. Thefeed remaining in each pen was weighed and recorded on study days 17, 28and 35. The feed intake during days 0-17, 17-28, and 0-35 wascalculated.

Weight Gains and Feed Conversion

Average bird weight, on a pen basis, on each weigh day was summarized.Bird weight gain by pen days 17-28 was calculated. The average feedconversion was calculated on the study days 17 and 28 (i.e. days 0-17,17-35, and 0-35) using the total feed consumption for the pen divided bythe total weight of surviving birds. Adjusted feed conversion wascalculated using the total feed consumption in a pen divided by thetotal weight of surviving birds and weight of birds that died or wereremoved from that pen.

Coccidiosis Challenge

All birds will each receive a 1× dose of Coccivac by spray cabinet onapproximately study day 0.

Clostridium perfringens Challenge

Clostridium Challenge

The Clostridium perfringens culture (CL-15) was grown ˜5 hrs at ˜37° C.in Fluid

Thioglycollate medium containing starch. CL-15 is a field strain ofClostridium perfringens from a broiler outbreak in Colorado. For eachpen of birds, a fixed amount of the broth culture (˜2-3 ml/bird) wasmixed with a fixed amount of treatment feed (˜25 g/bird) in the feedertray. The amount of feed, volume and quantitation of culture inoculum,and number of days dosed were documented in the final report and allpens were treated the same. Birds will receive the C. perfringensculture for one day (Study day 17). The target is 10% mortality with aminimum 5% in the challenged, non-medicated group.

Method of Administration

Administration of the Clostridium perfringens (CL-15, Type A, a and (32toxins) cultures in this study was via the feed. Feed from each pen'sfeeder was used to mix with the culture. Prior to placing the culturesin the pens the treatment feed was removed from the birds forapproximately 4-8 hours. For each pen of birds, a fixed amount (˜2.5ml/bird) of the broth culture at a concentration of approximately2.0-9.0×10⁸ cfu/ml was mixed with a fixed amount of feed (˜25 g/bird) inthe feeder tray and all challenged pens were treated the same. Most ofthe culture-feed was consumed within 1-2 hours. So that birds in alltreatments are treated similar, the groups that are not challenged willalso have the feed removed during the same time period as the challengedgroups.

Lesion Scoring

On study day 21, 5 birds were randomly selected from each pen (by firstbird caught), sacrificed and evaluated for intestinal lesions scored fornecrotic enteritis. Lesions were scored as follows:

0=normal: no NE lesions, small intestine has normal elasticity (rollsback to normal position after being opened)

1=mild: small intestinal wall was thin and flaccid (remains flat whenopened and doesn't roll back into normal position after being opened);excess mucus covering mucus membrane

2=moderate: noticeable reddening and swelling of the intestinal wall;minor ulceration and necrosis of the intestine membrane; excess mucus

3=severe: extensive area(s) of necrosis and ulceration of the smallintestinal membrane; significant hemorrhage; layer of fibrin andnecrotic debris on the mucus membrane (Turkish towel appearance)

4=dead or moribund: bird that would likely die within 24 hours and hasNE lesion score of 2 or more; or birds that died due to necroticenteritis.

TABLE 41 Results Avg Individual Adj. Feed NE- Lesion Trt Group Bird WtGain (kg) Conversion Mortality Score 1 1.912 1.735 39.5% 3.55 2 2.0901.712   5% 1.00 3 1.968 1.724 27.5% 3.4 4 2.006 1.695 24.8% 2.9

The birds were treated with a composition of Ascus microorganisms todetermine their effects on performance and the prevention of Clostridiumperfrigens infection. Two different microbial compositions were tested.The first composition consisted of Ascusbbr_4729, Ascusbbr_331885,Ascusbbr_170211, and the second consisted of Ascusbbr_4729, Ascusbbr_33,Ascusbbr_127. Microorganisms were administered once to the experimentalbirds via spray application prior to pen placement. All birds were on acommercially relevant pelleted feed.

Birds were challenged with C. perfringens on day 17 of the study. On day21, 5 birds were randomly selected, sacrified, and lesion scored.Mortality and feed intake were measured throughout the experiment. Atthe end of the experiment, birds were sacrificed and weighed. Feedconversion was calculated based on the total feed consumption for thepen divided by the total weight of the surviving birds. The treatmentgroup receiving Ascus microorganism composition 1 (treatment 3) wasfound to have slightly improved feed conversion (0.63%), slightly higherweight (2.93%), slightly lower lesion scores (4.23%), and lower percentmortality (30.37%) when compared to the challenged control. Thetreatment group receiving Ascus microorganism composition 2 (treatment4) was found to have improved feed conversion (2.31%), higher weight(4.91%), lower lesion scores (18.31%), and lower percent mortality(37.22%) when compared to the challenged control. The salinomycincontrol outperformed all groups.

Example VII. TRIAL 4—In Vivo Evaluation of Necrotic Enteritis withMultiple Ascus Microbial Compositions vs. Clostridium perfringens

Experimental Design

Experimental Ration

Rations consisted of non-medicated commercial-type broiler starter,grower, and finisher diets compounded according to NRC guidelines andcontained feedstuffs commonly used in the United States. Rations werefed ad libitum from date of chick arrival as follows: Starter—DOT 0until DOT 21, grower DOT 21 to DOT 35, and finisher DOT 35 to DOT 42(study termination). Diets were fed as crumbles (starter feed) orpellets (grower and finisher feed).

Animal Information

One thousand eight hundred (1,800) day-of-hatch Cobb male broiler chickswere obtained. The strain was Cobb×Cobb. Birds were sexed at thehatchery. All birds were vaccinated by spray cabinet with a commercialcoccidia vaccine at recommended dosage. Only healthy appearing chickswere used in this study.

Housing

Upon arrival chicks were raised in 5×10 feet floor pens (stockingdensity of 1.0 feet per bird) with approximately four (4) inches offresh pine shavings (at placement), in a solid-sided barn, with concretefloors under ambient humidity. Litter was not replaced or amended duringthe course of this study. Feed and water were available ad libitumthroughout the trial. Each pen contained 1 (one) tube feeder and 1 (one)bell drinker (50 bird to feeder/drinker ratio). Thermostaticallycontrolled gas heaters were the primary heat source for the barn (ifneeded). One (1) heat lamp per pen provided supplemental heat duringbrooding. Fans were used to cool birds. Birds were provided a lightingprogram as per the primary breeder recommendations. The pen diagram wasdocumented and included in final report with source data.

Probiotic Administration

After coccidia vaccine administrations all chicks in Treatments 3 werecoarse sprayed with 0.25 ml/chick of the Ascus probiotic and placedunder bright light to allow preening. Once dry, chicks were placed intreatment appropriate pens.

TABLE 42 Treatments CLOSTRIDIUM TREATMENT COCCI- PERFRINGENS PENS/BIRDS/ ID DESCRIPTION VACCINE CHALLENGE ¹ TREATMENT PEN T1 NonmedicatedDOT 0 DOT 19, 12 50 Cocci Vaccine 20, & 21 (Challenge Control) T2Nonmedicated DOT 0 NO 12 50 Cocci Vaccine (Negative Control) T3 AscusProbiotic: DOT 0 DOT 19, 12 50 Ascusbbr_409, 20, & 21 Ascusbbr_5796,Ascusbbr_1686 T4 Ascus Probiotic: DOT 0 DOT 19, 12 50 Ascusbbr_409, 20,& 21 Ascusbbr_5796, Ascusbbr_185064 ¹ DOT 19, 20, & 21: Clostridiumperfringens was added into the water at a dose of approximately 1 × 10⁸CFU/ml/bird. DOT = Day of trial *Probiotics were sprayed on chicks at0.25 ml/chick for Treatments 3 and 4 at 1 day of age prior to placement

Bird Allocation and Pen Randomization

One thousand eight hundred birds were assigned to three treatment groupswith twelve replicate pens per treatment and 50 birds per pen. Penfacility was divided into twelve blocks with each block containing eachof the three treatment groups. Treatment groups were assigned to pensusing randomized complete block. The study began when birds were placed(day-of-hatch; DOT 0), at which time birds were allocated toexperimental pens. Only healthy birds were selected. On DOT 0, groupbody weights were recorded by pen. No birds were replaced during thecourse of the study.

Challenge Administration and Sample Collection and Analysis

Necrotic Enteritis Challenge

The challenge model consisted of coccidia from the DOT 0 vaccine, onecoccidia seeding at DOT 14, and Clostridium perfringens combination.

Clostridium perfringens In Drinking Water

Treatment feed and water was withdrawn for a few hours prior toadministration of Clostridium perfringens. A measured amount ofClostridium perfringens was added to water that was consumed within 30minutes was used for each pen. The Clostridium perfringens culture wasadded to this water and thoroughly mixed and given to birds in eachchallenge pen. Once the challenge water was consumed treatment feed andwater were returned to pen. Clostridium perfringens was added to thewater on DOT 19, 20, and 21 to all bird except for Birds in TreatmentGroup 2.

Coccidia Challenge

Was from cycling of DOT 0 vaccination and E. maxima (20 ml per pen)spread around feeders and drinkers on DOT 14. (Except Treatment Group2).

Necrotic Enteritis Lesion Scoring

On DOT 21 three (3) birds per pen were humanely euthanized, necropsiedand lesion scored.

Lesion score 0=Normal

Lesion score 1=Slight mucus covering small intestine

Lesion score 2=Necrotic small intestine mucosa

Lesion score 3=Sloughed and blood small intestine mucosa and contents

Intestinal Content Sampling

On DOT 21 and 42 collected samples of the small intestines of two (2)birds per pen.

Feed Changes

Birds received treatment appropriate feed from DOT 0 to DOT 42. On DOT21 remaining starter feed was removed, weighed, and replaced with growerfeed. On DOT 35 remaining grower feed was removed, weighed, and replacedwith finisher feed. On DOT 42 remaining finisher feed was removed andweighed back. All unconsumed feed was weighed and disposed of in theSPRG onsite disposal pit.

Body and Feed Weight

All birds were weighed by pen on DOT 0, 21, 35, and 42. Feed added toeach pen's feeder was weighed at the beginning of each formulationperiod on DOT 0, 20, and 35 (starter, grower, and finisherrespectively). Any additional bags of feed were weighed (and documented)for each pen (as required) during each formulation period. Feed wasdistributed as needed to feeders from pre-weighed bags (assigned to eachpen) throughout each period. Feed remaining in feeders (and feed bags ifapplicable) was weighed and disposed of on DOT 21, 35, and 42. Empty penfeeder weights were recorded prior to study initiation. The trial wasterminated on DOT 42.

Management

Disease Control

No concomitant drug therapy was used during the study. Disposableplastic boot were worn by all study personnel required to enter pens(e.g., collect birds for study procedures). The disposable plastic bootswere removed as the person stepped out of pen to avoid tracking fecalmaterial throughout the facility. Disposable plastic boots were properlydisposed of after use.

Monitoring

All birds were monitored for general flock condition, temperature,lighting, water, feed, litter condition, and unanticipated houseconditions/events. Findings were documented twice daily during theregular working hours (one observation recorded final study day). OnSaturday, Sunday, and observed holidays, one (1) observation wasrecorded.

Mortality

Pens were checked daily for mortality. Birds were only culled to relievesuffering. Date and removal weight (kg) were recorded on all birdsculled (or found dead). A gross necropsy was performed on all dead orculled birds to determine the bird sex and probable cause of death.Signs of Necrotic Enteritis or non-specific enteritis were noted.

Bird and Feed Disposition

All birds were disposed of by appropriate methods. All mortalities andremaining feeds (including mixer flushes) were buried in the SouthernPoultry Research Group on site disposal pit.

Scales

Scale maintenance and standardization procedures were followed prior touse.

Source Data Control and Handling

Data were recorded in indelible ink. Entries were legible and sourcedata sheet signed (or initialed), and dated by individual recordingentry. All source data errors and/or changes were initialed, dated, anda brief explanation (or error code) written directly on form.

Data Management

Data management and statistical analysis of weight gain, feedconsumption, and feed conversion, and lesion score results wereperformed.

TABLE 43 Results Avg Individual Adj. Feed NE- Lesion Trt Group Bird WtGain (kg) Conversion Mortality Score 1 2.607 1.709 15.8%  0.5 2 2.5161.698 5.2%  0 3 2.518 1.690 10% 0.15 4 2.559 1.694 12% 0.4

The birds were treated with a composition of Ascus microorganisms todetermine their effects on performance and the prevention of Clostridiumperfrigens infection. Two different microbial compositions were tested.The first composition consisted of Ascusbbr_409, Ascusbbr_5796,Ascusbbr_1686, and the second consisted of Ascusbbr_409, Ascusbbr_5796,and Ascusbbr_185064. Microorganisms were administered once to theexperimental birds via spray application prior to pen placement. Allbirds were on a commercially relevant pelleted feed.

Birds were challenged with C. perfringens on day 17 of the study. On day21, 5 birds were randomly selected, sacrified, and lesion scored.Mortality and feed intake were measured throughout the experiment. Atthe end of the experiment, birds were sacrificed and weighed. Feedconversion was calculated based on the total feed consumption for thepen divided by the total weight of the surviving birds. The treatmentgroup receiving Ascus microorganism composition 1 (treatment 3) wasfound to have slightly improved feed conversion (1.11%), lower lesionscores (70.0%), and lower percent mortality (36.70%) when compared tothe challenged control. The treatment group receiving Ascusmicroorganism composition 2 (treatment 4) was found to have slightlyimproved feed conversion (0.88%), lower lesion scores (20.0%), and lowerpercent mortality (24.05%) when compared to the challenged control.Surprisingly, the challenged control exhibited the highest weight gain.

Example VIII. Comparative Analysis of MIC Scores from Published Work ofOther Groups

Utilizing Ascus Biosciences' technology, the performance of currentlyavailable microbial feed additive products was predicted.

Direct-fed microbial products that claim to enhance broiler performanceare available on the market. A few of these products containmicroorganism strains that are native chicken gastrointestinalmicroorganisms or are within 97% sequence similarity of nativegastrointestinal microorganisms. Here, we've identified the strains thatare used in these products, and calculated their platform score withrespect to feed efficiency and body weight (FIG. 15 and FIG. 16). As canbe seen from the curves, many of the currently available strains fallbelow the threshold used to define “useful” and “non-useful” strains.The one strain above the cutoff, Enterococcus faecium, has shownbeneficial effects when fed to broiler chickens.

Other common strains used in fowl/poultry direct fed microbial products,were either not found in the gastrointestinal tract of any birds or wereless than 97% similar to a strain found within the birds. Scores couldnot be generated for these microorganisms (Table 44).

TABLE 44 Microbes not appearing on the curve in FIG. 15 or FIG. 16.Microbial Organism Bacillus subtilis DSM 29870 Bacillus subtilis DSM29871 Bacillus subtilis AJ276351 Bacillus vallimortis AB021198 Bacillusamyloliquefaciens DSM 29869 Bacillus amyloliquefaciens DSM 29872Bifidobacterium animalis

Enterococcus faecium: 0.72083

Positive effects on overall weight gain, did not change FCR: Effects ofdietary Enterococcus faecium on growth performance, carcasscharacteristics, faecal microbiota, and blood profile in broilers. doi:10.17221/8680-VETMED

Positive effects of weight gain: Effects of Enterococcus faeciumsupplementation and floor type on performance, morphology oferythrocytes and intestinal microbiota in broiler chickens. doi:10.1080/00071668.2010.507241.

Positive effects on weight gain: Effects of Enterococcus faecium anddried whey on broiler performance, gut histomorphology and intestinalmicrobiota. DOI: 10.1080/17450390601106655

Positive effects on weight gain and intestinal development: IntestinalStructure and Function of Broiler Chickens on Diets Supplemented with aSynbiotic Containing Enterococcus faecium and Oligosaccharides.doi:10.3390/ij ms9112205.

Pediococcus acidilactici: 0.17931

Did not affect body weight: Effects of dietary probiotic (Pediococcusacidilactici) supplementation on performance, nutrient digestibility,egg traits, egg yolk cholesterol, and fatty acid profile in laying hens.DOI: doi.org/10.3382/ps.2012-02370.

Did not affect body weight: Efficacy of Bactocell® and Toyocerin® asProbiotics on Growth Performance, Blood Parameters and IntestinalMorphometry of Turkey Poults.

No significant difference to performance: Growth performance and immuneresponse of broiler chickens fed diets supplemented with probiotic and(or) prebiotic preparations.

Probiotic of P. acidilactici alone did not improve performance: Effectof Probiotic, Prebiotic, and Synbiotic on Broiler Performance.

Lactobacillus salivarius DSM 16351: 016462 (weight), 0.31742 (feedconversion)

Does not improve body weight, slight effect on feed conversion ratio attimes: Influence of probiotic administration by feed or water on growthparameters of broilers reared on medicated and nonmedicated diets. DOI:doi.org/10.3382/japr.2009-00084

Lactobacillus reuteri: 0.26096

Slight effect on feed conversion ratio at times: Influence of probioticadministration by feed or water on growth parameters of broilers rearedon medicated and nonmedicated diets. DOI: doi.org/10.3382/japr.2009-00084.

Bacillus amyloliquefaciens AB255669: 0.18434

No effect on performance: Efficacy of protected sodium butyrate, aprotected blend of essential oils, their combination, and Bacillusamyloliquefaciens spore suspension against artificially induced necroticenteritis in broilers DOI: https://doi.org/10.3382/ps.2011-01853.

Example IX. Volatile Fatty Acid and Carbon Source Assays

Volatile Fatty Acid Assay

In order to assess the ability of the strains to produce volatile fattyacids, HPLC was utilized to measure the concentrations of acetic acid,butyric acid, propionic acid, and lactic acid in spent media.

A single colony was picked from each of the desired strains (fromanaerobic agar plates) and was inoculated into fresh media. At the sametime, a media blank was also prepared. The cultures and the media blankwere incubated at 37° C. until significant growth was visible (˜5 days).The OD600 was determined for each culture, and the strain ID wasconfirmed with Illumina sequence. An aliquot of culture was filtersterilized into an acid washed and autoclaved glass 15 mL sample vialwhich was then analyzed by HPLC.

HPLC reactions were performed on a BioRad Aminex HPX-87H with thefollowing conditions: 60° C., 0.5 mL/min mobile phase 0.00325 N H2S04,500 psi, 35C RI detector, 45 min run time, injection volume of 5 μL.Concentrations of acetic acid, butyric acid, propionic acid, and lacticacid were quantified for the medium blanks as well as the sterilefiltered culture samples. The strains were considered positive forvolatile fatty acid production if the detected concentration of theindividual fatty acids in the spent medium were higher than in the mediablank. See Table 45.

TABLE 45 VFA production from microbes of the present disclosure. lacticacetic propionic butyric Strain ID Condition/Media acid acid acid acidAscusbbr_94 MRS + 0 0 0 Ascusbbr_94 MRS 10 mM_Acetic_Acid + − 0 0Ascusbbr_91 M2GSC Salts Butyric Acid 0 + + − Ascusbbr_91 BL Amino AcidD + + 0 0 Ascusbbr_91 CMC Amino Acid D 0 + + 0 Ascusbbr_84 CMC AminoAcid D 0 + + 0 Ascusbbr_830 BL Amino Acid D + + 0 0 Ascusbbr_830 CMCAmino Acid D 0 + + 0 Ascusbbr_7779 M2GSC_Arabinose Xylose + + + +Ascusbbr_7363 Spirillium Butyric Acid − 0 0 + Ascusbbr_7363 M2GSC SaltsButyric Acid 0 + + − Ascusbbr_72076 1:10 MRS + + 0 0 Ascusbbr_72076BL + + + 0 Ascusbbr_72076 M2GSC_Arabinose Xylose + + + + Ascusbbr_72076BL Amino Acid D + + 0 0 Ascusbbr_6957 BL Amino Acid D + + 0 0Ascusbbr_6957 CMC Amino Acid D 0 + + 0 Ascusbbr_6097 M2GSC_ArabinoseXylose + + + + Ascusbbr_6097 Spirillium Butyric Acid − 0 0 +Ascusbbr_6097 BL Amino Acid D + + 0 0 Ascusbbr_6097 CMC Amino Acid D0 + + 0 Ascusbbr_5796A MRS + + 0 0 Ascusbbr_5796B MRS + + 0 0Ascusbbr_5796C MRS + + 0 0 Ascusbbr_5796A BL + + 0 0 Ascusbbr_5796BBL + + 0 0 Ascusbbr_5796C BL + + + 0 Ascusbbr_5796A 1:10 MRS + + 0 0Ascusbbr_5796A M2GSC_Arabinose Xylose + + + + Ascusbbr_5796A BL AminoAcid D + + 0 0 Ascusbbr_5796A CMC Amino Acid D 0 + + 0 Ascusbbr_5796A1:10 MRS 0 + 0 0 Ascusbbr_48584 MRS + + 0 0 Ascusbbr_4729M2GSC_Arabinose Xylose + + + + Ascusbbr_42760A MRS + + 0 0Ascusbbr_42760A 1:10 MRS + + 0 0 Ascusbbr_42760B 1:10 MRS 0 + 0 0Ascusbbr_42760A BL + + + 0 Ascusbbr_42760A M2GSC_ArabinoseXylose + + + + Ascusbbr_42760A BL Amino Acid D + + 0 0 Ascusbbr_409AMRS + + 0 0 Ascusbbr_409B MRS + + 0 0 Ascusbbr_409A BL + + + 0Ascusbbr_409B BL + + + 0 Ascusbbr_409C BL + + + 0 Ascusbbr_409AM2GSC_Arabinose Xylose + + + + Ascusbbr_409A BL Amino Acid D + + 0 0Ascusbbr_409B BL Amino Acid D + + 0 0 Ascusbbr_409C BL Amino Acid D + +0 0 Ascusbbr_409A CMC Amino Acid D 0 + + 0 Ascusbbr_409B CMC Amino AcidD 0 + + 0 Ascusbbr_409A 1:10 MRS 0 + 0 0 Ascusbbr_38717A MRS + + 0 0Ascusbbr_38717B MRS + + 0 0 Ascusbbr_38717A BL + + + 0 Ascusbbr_38717AM2GSC_Arabinose Xylose + + + + Ascusbbr_38717A 1:10 MRS 0 + 0 0Ascusbbr_36257 BL Amino Acid D + + 0 0 Ascusbbr_359892 CMC Amino Acid D0 + + 0 Ascusbbr_35 M2GSC_Arabinose Xylose + + + + Ascusbbr_339Spirillium Butyric Acid − 0 0 + Ascusbbr_339 M2GSC Salts Butyric Acid0 + + − Ascusbbr_339 CMC Amino Acid D 0 + + 0 Ascusbbr_331885 MRS + + 00 Ascusbbr_331885 BL Amino Acid D + + 0 0 Ascusbbr_33 MRS + + + 0Ascusbbr_33 BL + + 0 0 Ascusbbr_33 1:10 MRS 0 + 0 0 Ascusbbr_32731A MRS− + 0 0 Ascusbbr_32731A M2GSC Salts Butyric Acid 0 + + − Ascusbbr_32731ABL Amino Acid D + + 0 0 Ascusbbr_32731A CMC Amino Acid D 0 + + 0Ascusbbr_32731A 1:10 MRS + + 0 0 Ascusbbr_32731B 1:10 MRS 0 + 0 0Ascusbbr_322104 1:10 MRS 0 + 0 0 Ascusbbr_313454 M2GSC_ArabinoseXylose + + + + Ascusbbr_31 BL Amino Acid D + + 0 0 Ascusbbr_3089Spirillium Butyric Acid − 0 0 + Ascusbbr_285160 M2GSC_ArabinoseXylose + + + + Ascusbbr_285160 BL Amino Acid D + + 0 0 Ascusbbr_2851601:10 MRS 0 + 0 0 Ascusbbr_28 Spirillium Butyric Acid − 0 0 + Ascusbbr_28M2GSC Salts Butyric Acid 0 + + − Ascusbbr_28 BL Amino Acid D + + 0 0Ascusbbr_28 CMC Amino Acid D 0 + + 0 Ascusbbr_265A 1:10 MRS + + 0 0Ascusbbr_265B 1:10 MRS 0 + 0 0 Ascusbbr_265A BL + + + 0 Ascusbbr_265AM2GSC_Arabinose Xylose + + + + Ascusbbr_25200 BL + + + 0 Ascusbbr_25200M2GSC_Arabinose Xylose + + + + Ascusbbr_247 M2GSC_ArabinoseXylose + + + + Ascusbbr_247A M2GSC Salts Butyric Acid 0 + + −Ascusbbr_247B M2GSC Salts Butyric Acid 0 + + − Ascusbbr_247A BL AminoAcid D + + 0 0 Ascusbbr_247A CMC Amino Acid D 0 + + 0 Ascusbbr_247B CMCAmino Acid D 0 + + 0 Ascusbbr_2158 CMC Amino Acid D 0 + + 0Ascusbbr_21169 MRS + + 0 0 Ascusbbr_21169 BL + + 0 0 Ascusbbr_19 MRS + 00 0 Ascusbbr_19 1:10 MRS + + 0 0 Ascusbbr_185064 MRS − + + 0Ascusbbr_185064 BL Amino Acid D + + 0 0 Ascusbbr_1789 Spirillium ButyricAcid − 0 0 + Ascusbbr_1789 BL Amino Acid D + + 0 0 Ascusbbr_1789 CMCAmino Acid D 0 + + 0 Ascusbbr_1789 1:10 MRS 0 + 0 0 Ascusbbr_173Spirillium Butyric Acid − 0 0 + Ascusbbr_173 BL Amino Acid D + + 0 0Ascusbbr_173 CMC Amino Acid D 0 + + 0 Ascusbbr_17 M2GSC_ArabinoseXylose + + + + Ascusbbr_1686 MRS + + 0 0 Ascusbbr_1686 BL + + + 0Ascusbbr_1686 MRS 10 mM_Acetic_Acid + − 0 0 Ascusbbr_1686M2GSC_Arabinose Xylose + + + + Ascusbbr_1686 Spirillium Butyric Acid − 00 + Ascusbbr_1686 BL Amino Acid D + + 0 0 Ascusbbr_1686 CMC Amino Acid D0 + + 0 Ascusbbr_1686 1:10 MRS 0 + 0 0 Ascusbbr_14834 1:10 MRS + + 0 0Ascusbbr_14834 BL + + + 0 Ascusbbr_14834 Spirillium Butyric Acid − 0 0 +Ascusbbr_14834 M2GSC Salts Butyric Acid 0 + + − Ascusbbr_14834 BL AminoAcid D + + 0 0 Ascusbbr_14834 CMC Amino Acid D 0 + + 0 Ascusbbr_14690AMRS + 0 0 0 Ascusbbr_14690B MRS + 0 0 0 Ascusbbr_14690C MRS + 0 0 0Ascusbbr_14690A M2GSC_Arabinose Xylose + + + + Ascusbbr_14690ASpirillium Butyric Acid − 0 0 + Ascusbbr_14690A BL Amino Acid D + + 0 0Ascusbbr_14690A 1:10 MRS 0 + 0 0 Ascusbbr_144 BL Amino Acid D + + 0 0Ascusbbr_1436 MRS + + 0 0 Ascusbbr_1436A 1:10 MRS + + 0 0 Ascusbbr_1436B1:10 MRS 0 + 0 0 Ascusbbr_1436A BL + + + 0 Ascusbbr_1436AM2GSC_Arabinose Xylose + + + + Ascusbbr_1436A Spirillium Butyric Acid −0 0 + Ascusbbr_1436A M2GSC Salts Butyric Acid 0 + + − Ascusbbr_1436A BLAmino Acid D + + 0 0 Ascusbbr_1436A CMC Amino Acid D 0 + + 0Ascusbbr_136 Spirillium Butyric Acid − 0 0 + Ascusbbr_136 M2GSC SaltsButyric Acid 0 + + − Ascusbbr_13398 CMC Amino Acid D 0 + + 0Ascusbbr_128 M2GSC_Arabinose Xylose + + + + Ascusbbr_127 MRS + + 0 0Ascusbbr_10593A MRS + + 0 0 Ascusbbr_10593B MRS + + 0 0 Ascusbbr_10593ASpirillium Butyric Acid − 0 0 + Ascusbbr_10593A M2GSC Salts Butyric Acid0 + + − Ascusbbr_10593A BL Amino Acid D + + 0 0

Soluble Carbon Source Assay

In order to assess the ability of the strains to degrade various solublecarbon sources, OD600 was used to measure growth of strains onparticular carbon sources over a period of time.

A single colony from each of the desired strains (on anaerobic agarplates) was inoculated into fresh medium. Strains were inoculated into acarbon source assay anaerobically; the assay was set up in a 2 mLsterile 96 well plate, with each well containing M2GSC salts, vitamins,minerals, sodium sulfide, and a single carbon source. Carbon sourcesincluded whole chicken feed, Soytone, Maltose, Raffinose, Starch,Arabinose, Sucrose, Xylose, Succinate, Cellobiose, Casamino acids,Glucose, Galactose, Manitol, Peptone, Gluconate, Malt Extract, CaseinDigest, Beef Extract, and Chitosan. Cells were inoculated such that eachwell started at an OD600 of 0.01. The ODs were read at 600 nm with the“Synergy H4 hybrid plate reader”. Strain ID was confirmed with Illuminasequencing after all wells were in stationary phase.

XTT reduction was simultaneously measured by adding 100 ul of carbonsource with strain culture to a 200 ul flat bottom plate. To thisaliquot 50 ul of the XTT mix (5 ml of sterile XTT with 100 ul of sterileN-methyl dibenzopyrazine methyl sulfate) was added. This culture wasthen incubated for 1 hour at 37° C. anaerobically in the dark. XTTreduction was determined by absorbance at 475 nm subtracted for thenon-specific absorbance at 660 nm as well as the appropriate media andstrain blanks. See Table 46.

TABLE 46 Carbon source growth assays with microbes of the presentdisclosure. Whole Chicken Feed Soytone Maltose Raffinose StarchArabinose Sucrose Xylose Succinate Cellobiose CasAmino Ascusbbr_94 + +Ascusbbr_91 + + + Ascusbbr_84 + + + Ascusbbr_830 + + +Ascusbbr_7779 + + + + Ascusbbr_7363 + + + Ascusbbr_72076 + + + +Ascusbbr_6957 + + + Ascusbbr_6097 + + + + + +Ascusbbr_5796 + + + + + + + Ascusbbr_48584 + Ascusbbr_4729 + + + +Ascusbbr_42760 + + + + Ascusbbr_409 + + + + + + Ascusbbr_38717 + + + + +Ascusbbr_36257 Ascusbbr_359892 + + + Ascusbbr_35 + + + +Ascusbbr_339 + + + + Ascusbbr_331885 Ascusbbr_33 + +Ascusbbr_32731 + + + Ascusbbr_322104 Ascusbbr_313454 + + + + Ascusbbr_31Ascusbbr_3089 + Ascusbbr_285160 + + + + Ascusbbr_28 + + + +Ascusbbr_265 + + + + Ascusbbr_25200 + + + + Ascusbbr_247 + + + + +Ascusbbr_2158 + + + Ascusbbr_21169 Ascusbbr_19 Ascusbbr_185064 + + + +Ascusbbr_1789 + + + + Ascusbbr_173 + + + + Ascusbbr_17 + + + +Ascusbbr_1686 + + + + + + Ascusbbr_14834 + + + +Ascusbbr_14690 + + + + + Ascusbbr_144 Ascusbbr_1436 + + + + + + + +Ascusbbr_136 + + + Ascusbbr_13398 + + + Ascusbbr_128 + + + +Ascusbbr_127 + Ascusbbr_10593 + + + Malt Casein Beef Glucose GalactoseManitol Peptone Gluconate Extract Digest Extract Chitosan MRSAscusbbr_94 + + + + Ascusbbr_91 + + + Ascusbbr_84 + + +Ascusbbr_830 + + + Ascusbbr_7779 + + Ascusbbr_7363 + + +Ascusbbr_72076 + + + + + Ascusbbr_6957 + + + Ascusbbr_6097 + + +Ascusbbr_5796 + + + + + + Ascusbbr_48584 + + + + + + + + + +Ascusbbr_4729 + + Ascusbbr_42760 + + + + + Ascusbbr_409 + + + + +Ascusbbr_38717 + + + + + + + + + Ascusbbr_36257 + +Ascusbbr_359892 + + + Ascusbbr_35 + + Ascusbbr_339 + + +Ascusbbr_331885 + + + + Ascusbbr_33 + + + + Ascusbbr_32731 + + + + +Ascusbbr_322104 + + + + Ascusbbr_313454 + + Ascusbbr_31 + +Ascusbbr_3089 + Ascusbbr_285160 + + + + + Ascusbbr_28 + + +Ascusbbr_265 + + + + + Ascusbbr_25200 + + + Ascusbbr_247 + + +Ascusbbr_2158 + + + Ascusbbr_21169 + + + Ascusbbr_19 + + + +Ascusbbr_185064 + + + + + Ascusbbr_1789 + + + + + Ascusbbr_173 + + +Ascusbbr_17 + + Ascusbbr_1686 + + + + + Ascusbbr_14834 + + + + +Ascusbbr_14690 + + + + + Ascusbbr_144 + + Ascusbbr_1436 + + + + +Ascusbbr_136 + + + Ascusbbr_13398 + + + Ascusbbr_128 + + Ascusbbr_127 +Ascusbbr_10593 + + +

Insoluble Carbon Source Assay

In order to assess the ability of the strains to degrade insolublecarbon sources, visual inspection was leveraged to qualitativelydetermine a strain's degradation capabilities.

For pure cultures, a single colony from each of the desired strains(from anaerobic agar plates) was inoculated into anaerobic Hungate tubescontaining Lowe's semi defined media with cellulose paper, starch, orgrass as the sole carbon source. (Lowe et al. 1985. J. Gen. Microbiol.131:2225-2229). A medium blank was also prepared. Cultures were checkedvisually for degradation of insoluble carbon sources. See FIG. 10.

Enrichments

The same protocols as described above for the VFA assay and the solublecarbon source assay were used for enrichment assays, but instead ofinoculating with a single colony, fresh gastrointestinal sample wasused. Gastrointestinal sample inocula and enrichments were Illuminasequenced to determine presence or absence of target strains. Sequencingdatasets were integrated with cell count data to determine if targetstrains grew in vitro.

Example X. Competitive Exclusion Assays (In Vitro)

In order to assess the ability of the strains to compete againstpathogens in the gastrointestinal tract of the fowl, competitiveexclusion against Clostridium perfringens or Salmonella enterica wasmeasured by co-culturing strains together in a medium representative ofthe broiler GI tract as well as in a minimal salts medium. Aftersubstantial cell growth, each co-culture was sequenced. The relativeabundance of each strain was then used to determine the efficacy of thestrains at competing with or inhibiting the pathogen.

Single colonies of the strains and pathogens were inoculated into 500 μLof MRS and TSB anaerobically. The OD readings were measured thefollowing day, and fresh MRS and TSB co-cultures were inoculated suchthat each strain was at a starting OD of 0.01. 300 μL of the startinginoculum was collected and sequenced to provide a relative abundance atTO, and the starting abundance of each strain was confirmed.

Strains were considered successful at competing against the pathogenicstrains if the pathogen experienced a theoretical percent decrease(relative abundance as determined by sequencing*coculture OD) of atleast 50% when compared to the starting inoculum. Strains wereconsidered to have a weak competitive exclusion effect if the final ODof the co-culture was lower than the OD of the pure pathogen culture.Strains were considered negative if they were overgrown by the pathogen.See Table 47 for results. When cocultured with Clostridium perfringens,15 of the 24 strains (62.5%) exhibited an inhibitory effect against C.perfrigens. Of the strains cocultured with Salmonella enterica, 4 of the7 strains (57.14%) tested exhibited an exhibitory effect against S.enterica. Strains that shared similar 16S sequences (97% sequencesimilarity) tended to exhibit similar effects on the pathogens—thisincludes Ascusbbr_33A and B; Ascusbbr_409A and B; Ascusbbr_5796A, B, andC; Ascusbbr_14690A, B, and C; and Ascusbbr_38717A and B. Strains relatedto Ascusbbr_10593, however, did show have a few differing results.Ascusbbr_10593A and B both inhibited S. enterica, but Ascusbbr_10593Aseemed to have a slightly stronger inhibitory effect.

TABLE 47 Strain competition data for C. perfringens and S. enterica.Clostridium Salmonella perfringens enterica Ascusbbr_19 − NTAscusbbr_33A − NT Ascusbbr_33B − NT Ascusbbr_127 − NT Ascusbbr_409A ++NT Ascusbbr_409B ++ NT Ascusbbr_1436 ++ + Ascusbbr_1686 ++ NTAscusbbr_5796A ++ NT Ascusbbr_5796B ++ NT Ascusbbr_5796C ++ NTAscusbbr_21169 ++ NT Ascusbbr_38717A ++ NT Ascusbbr_38717B ++ NTAscusbbr_10593A + ++ Ascusbbr_10593B + + Ascusbbr_14690A − NTAscusbbr_14690B − NT Ascusbbr_14690C − − Ascusbbr_32731 − −Ascusbbr_42760 + ++ Ascusbbr_48584 ++ NT Ascusbbr_185064 − NTAscusbbr_331885 ++ − Key − = Strain was outcompeted by the pathogen + =Strain weakly inhibited the pathogen ++ = Strain inhibited the pathogenNT = Condition not tested

NUMBERED EMBODIMENTS OF THE DISCLOSURE

1. A microbial composition comprising at least one microbial strainselected from Table 1 and/or Table 2.2. A microbial composition comprising at least one microbial strain,wherein the at least one microbial strain comprises a 16S rRNA sequenceselected from SEQ ID NOs:1-50, or SEQ Nos:338-364; or an ITS sequenceselected from SEQ ID NOs: 51-58.3. The microbial composition of claim 2, wherein the at least onemicrobial strain comprises Ascusb_4729.4. The microbial composition of claim 2, wherein the at least onemicrobial strain comprises Ascusb_170211.5. The microbial composition of claim 2, wherein the at least onemicrobial strain comprises Ascusb_1686.6. The microbial composition of claim 2, wherein the at least onemicrobial strain comprises Ascusb_33. 7. The microbial composition ofclaim 2, wherein the at least one microbial strain comprises Ascusb_128.8. The microbial composition of claim 2, wherein the at least onemicrobial strain comprises Ascusb_4729 and Ascusb_170211.9. The microbial composition of claim 2, wherein the at least onemicrobial strain comprises Ascusb_4729, Ascusb_33, and Ascusb_313454.10. The microbial composition of any one of claims 1-9, wherein saidmicrobial composition is encapsulated.11. A composition comprising:

-   -   (a) a microbial composition of any one of claims 1-10, and    -   (b) an acceptable carrier.        12. The composition of claim 11, wherein the microbial        composition is encapsulated.        13. The composition of claim 11, wherein the encapsulated        microbial composition comprises a polymer selected from a        saccharide polymer, agar polymer, agarose polymer, protein        polymer, and lipid polymer.        14. The composition of claim 11, wherein the acceptable carrier        is selected from the group consisting of: edible feed grade        material, mineral mixture, water, glycol, molasses, and corn        oil.        15. The composition of claim 11, wherein the at least two        microbial strains forming the microbial consortium are present        in the composition at 10² to 10¹⁵ cells per gram of said        composition.        16. The composition of claim 11, wherein said composition is        mixed with animal feed. 17. A method of imparting at least one        improved trait upon an animal, said method comprising        administering the composition of claim 11 to said animal.        18. The method of claim 17, wherein said animal is a fowl. 19.        The method of claim 18, wherein said fowl is a broiler        chicken. 20. The method of claim 18, wherein the administration        comprises injecting the composition into one or more of the        crop, gizzard, cecum, small intestine, or large intestine of the        animal.        21. The method of claim 17, wherein said composition is        administered at least once per month.        22. The method of claim 21, wherein said composition is        administered at least once per week.        23. The method of claim 22, wherein said composition is        administered at least once per day. 24. The method of claim 17,        wherein the administration occurs each time the animal is fed.        25. The method of claim 17, wherein the administration is a        cloacal administration.        26. The method of claim 25, wherein the cloacal administration        comprises inserting a suppository comprising the composition        into the rectum of the animal.        27. The method of claim 17, wherein the administration is an        oral administration.        28. The method of claim 27, wherein the oral administration        comprises administering the composition in combination with the        animal's feed, water, litter, medicine, or vaccination.        29. The method of claim 27, wherein the oral administration        comprises applying the composition in a gel or viscous solution        to a body part of the animal, wherein the animal ingests the        composition.        30. The method of claim 17, wherein the administration comprises        spraying the composition onto the animal, and wherein the animal        ingests the composition.        31. The method of claim 17, wherein said at least one improved        trait is selected from the group consisting of: an increase in        weight; an increase in egg production; an increase of        musculature; an increase of vitamins in eggs; an increase of        fatty acid concentration in the gastrointestinal tract; and        increase in egg volume; an improved efficiency in feed        utilization and digestibility; an increase in polysaccharide and        lignin degradation; an increase in fat, starch, and/or protein        digestion; an increase in vitamin availability; an increase in        mineral availability; an increase in amino acid availability; pH        balance in the gastrointestinal tract; a reduction in methane        and/or nitrous oxide emissions; a reduction in manure        production; an improved efficiency of nitrogen utilization; an        improved efficiency of phosphorous utilization; an increased        resistance to colonization of pathogenic microbes that colonize        chickens; reduced mortality, increased production of        antimicrobials, increased clearance of pathogenic microbes,        increased resistance to colonization of pathogenic microbes that        infect chickens, increased resistance to colonization of        pathogenic microbes that infect humans; wherein said increase or        reduction is determined by comparing against an animal not        having been administered said composition.        32. The method of claim 31, wherein said increase in weight is        an increase by at least 1%.        33. The method of claim 31, wherein said reduction in manure        production is a reduction by at least 1%.        34. The method of claim 31, wherein said increase in        polysaccharide degradation is an increase in the degradation of        lignin, cellulose and/or hemicellulose.        35. The method of claim 31, wherein said increase in fatty acid        concentration is an increase in acetic acid, propionic acid,        and/or butyric acid.        36. The composition of claim 11, wherein the at least one        microbial strain exhibit an increased utility that is not        exhibited when said at least one microbial strain occurs alone,        or when said at least one microbial strain is present at        naturally occurring concentrations.        37. The composition of claim 11, wherein the at least one        microbial strain exhibits a synergistic effect on imparting at        least one improved trait in an animal.        38. A poultry feed supplement capable of increasing a desirable        phenotypic trait in a bird, the feed supplement comprising:    -   (a) a microbial consortium of any one of claims 1-9 present at a        concentration that does not occur naturally in said bird, and    -   (b) an acceptable carrier.        39. The poultry feed supplement of claim 38, wherein the        microbial consortium is encapsulated.        40. An isolated microbial strain selected from any one of the        microbial strains in Table 1 and/or Table 2.        41. An isolated microbial strain selected from the group        consisting of:    -   (a) Ascusb_4729 deposited as PATENT201703004    -   (b) Ascusb_170211 deposited as PATENT201703002    -   (c) Ascusb_1686 deposited as PTA-124016;    -   (d) Ascusb_33 deposited as B-67266;    -   (e) Ascusb_128 deposited as PATENT201703004;    -   (f) Ascusb_127 deposited as B-67265;    -   (g) Ascusb_14834 deposited as PTA-124016;    -   (h) Ascusb_313454 deposited as PATENT201703003;    -   (i) Ascusb_28 deposited as PTA-124039;    -   (j) Ascusb_144 deposited as PTA-124039;    -   (k) Ascusb_312 deposited as PATENT201703002; and    -   (l) Ascusb_2158 deposited as PTA-124039        42. An isolated microbial strain comprising a polynucleotide        sequence sharing at least 90% sequence identity with any one of        SEQ ID NOs:1-58 and 338-364.        43. A substantially pure culture of an isolated microbial strain        according to any one of claims 40 to 42.        44. A method of modulating the microbiome of a fowl, the method        comprising administering the composition of claim 12.        45. The method of claim 44, wherein the administration of the        composition imparts at least one improved trait upon the fowl.        46. The method of claim 45, wherein the at least one improved        trait is selected from the group consisting of: an increase in        weight; an increase in egg production; an increase of        musculature; an increase of vitamins in eggs; an increase of        fatty acid concentration in the gastrointestinal tract; and        increase in egg volume; an improved efficiency in feed        utilization and digestibility; an increase in polysaccharide and        lignin degradation; an increase in fat, starch, and/or protein        digestion; an increase in vitamin availability; an increase in        mineral availability; an increase in amino acid availability; pH        balance in the gastrointestinal tract; a reduction in methane        and/or nitrous oxide emissions; a reduction in manure        production; an improved efficiency of nitrogen utilization; an        improved efficiency of phosphorous utilization; an increased        resistance to colonization of pathogenic microbes that colonize        chickens; reduced mortality, increased production of        antimicrobials, increased clearance of pathogenic microbes,        increased resistance to colonization of pathogenic microbes that        infect chickens, increased resistance to colonization of        pathogenic microbes that infect humans; wherein said increase or        reduction is determined by comparing against an animal not        having been administered said composition.        47. The method of claim 46, wherein said increase in weight is        an increase by at least 1%.        48. The method of claim 46, wherein said reduction in manure        production is a reduction by at least 1%.        49. The method of claim 46, wherein said increase in        polysaccharide degradation is an increase in the degradation of        lignin, cellulose, and/or hemicellulose.        50. The method of claim 46, wherein said increase in fat        digestion, starch digestion, and/or protein digestion is an        increase by at least 1%.        51. The method of claim 46, wherein said increase in fatty acid        concentration is an increase in acetic acid, propionic acid,        and/or butyric acid.        52. The method of claim 45, wherein the modulation of the        microbiome is an increase in the proportion of the at least one        microbial strain of the microbiome, wherein the increase is        measured relative to a fowl that did not have the at least one        microbial strain administered.        53. The method of claim 45, wherein the modulation of the        microbiome is a decrease in the proportion of the microbial        strains present in the microbiome prior to the administration of        the composition, wherein the decrease is measured relative to        the microbiome of the fowl prior to the administration of the        composition.        54. A method of increasing resistance of poultry to the        colonization of pathogenic microbes, the method comprising the        administration of the composition of claim 11, wherein the        pathogen is unable to colonize the gastrointestinal tract of the        poultry.        55. The method of treating poultry for the presence of at least        one pathogenic microbe, the method comprising the administration        of the composition of claim 11.        56. The method of claim 55, wherein after administration of the        composition the relative abundance of the at least one        pathogenic microbe decreases to less than 5% relative abundance        in the gastrointestinal tract.        57. The method of claim 56, wherein the relative abundance of        the at least one pathogenic microbe decreases to least than 1%        relative abundance in the gastrointestinal tract.        58. The method of claim 56, wherein the at least one pathogenic        microbe is undetectable in the gastrointestinal tract.        59. The method of claim 58, wherein less than 10 days post        administration of the composition the at least one pathogenic        microbe is undetectable in the gastrointestinal tract.        60. The method of claim 58, wherein within 5-15 days post        administration of the composition the at least one pathogenic        microbe is undetectable in the gastrointestinal tract.        61. The method of claim 56, wherein the at least one pathogenic        microbe is also undetectable in or on eggs laid by the poultry.        62. The method of any one of claims 54-61, wherein the at least        one pathogenic microbe is selected from: Mycoplasma        gallisepticum, Mycoplasma meleagridis, Mycoplasma synoviae,        Pasteurella multocida, Clostridium perfringens, Clostridium        colinum, Clostridium botulinum, Salmonella typi, Salmonella        typhimurium, Salmonella enterica, Salmonella pullorum,        Salmonella gallinarum, Hemophilus gallinarum, Erysipelothrix        insidiosa, Campylobacter jejuni, Campylobacter coli,        Campylobacter lari, Listeria monocytogenes, Arcobacter butzleri,        Mycobacterium avium, and pathogenic strains of Escherichia coli        and Staphylococcus aureus.        63. The method of claim 62, wherein the at least one pathogenic        microbe is selected from Salmonella or Clostridium.        64. The composition of claim 11, wherein the microbial        composition comprises bacteria and/or fungi in spore form.        65. The composition of claim 11, wherein the microbial        composition comprises a dechlorinator and/or an oxygen        scavenger.

In aspects, the aforementioned microbial species—that is, a purifiedmicrobial population that comprises a bacteria with a 16S nucleic acidsequence, and/or a fungi with an ITS nucleic acid sequence, which is atleast about 97% identical to a nucleic acid sequence selected from thegroup consisting of: SEQ ID NOs: 1-387—are members of a Markush group,as the present disclosure illustrates that the members belong to a classof microbes characterized by various physical and functional attributes,which can include any of the following: a) the ability to convert acarbon source into a volatile fatty acid such as acetate, butyrate,propionate, or combinations thereof; b) the ability to degrade a solubleor insoluble carbon source; c) the ability to impart an increase inweight gain to fowl administered the microbe(s); d) the ability tomodulate the microbiome of the gastrointestinal tract of fowladministered the microbe; e) the ability to be formulated into ashelf-stable composition; f) the ability to exhibit a decrease in feedconversion ratio in fowl having been administered the microbe(s); g) theability to impart a decrease in pathogen-associated lesion formation inthe gastrointestinal tract; h) the ability to impart a decrease inpathogenic microbes in the gastrointestinal tract; and/or i) possessinga MIC score of at least about 0.2 if a bacteria and possessing a MICscore of at least about 0.2 if a fungi. Thus, the members of the Markushgroup possess at least one property in common, which can be responsiblefor their function in the claimed relationship.

As used herein “shelf-stable” refers to a functional attribute and newutility acquired by the microbes formulated according to the disclosure,which enable said microbes to exist in a useful/active state outside oftheir natural environment in the gastrointestinal tract (i.e. a markedlydifferent characteristic). Thus, shelf-stable is a functional attributecreated by the formulations/compositions of the disclosure and denotingthat the microbe formulated into a shelf-stable composition can existoutside the gastrointestinal tract and under ambient conditions for aperiod of time that can be determined depending upon the particularformulation utilized, but in general means that the microbes can beformulated to exist in a composition that is stable under ambientconditions for at least a few days and generally at least one week.Accordingly, a “shelf-stable fowl supplement” is a compositioncomprising one or more microbes of the disclosure, said microbesformulated in a composition, such that the composition is stable underambient conditions for at least one week, meaning that the microbescomprised in the composition (e.g. whole cell, spore, or lysed cell) areable to impart one or more beneficial phenotypic properties to a fowlwhen administered (e.g. increased weight gain, increased eggshelldensity, improved gastrointestinal health, and/or modulation of thegastrointestinal microbiome).

In some embodiments, the isolated microbial strains of the presentdisclosure further encompass mutants thereof. In some embodiments, thepresent disclosure further contemplates microbial strains having all ofthe identifying characteristics of the presently disclosed microbialstrains.

TABLE 45 Budapest Treaty Deposits of the Disclosure Depository AccessionNumber Date of Deposit ATCC PTA-124016 Mar. 2, 2017 ATCC PTA-124039 Mar.10, 2017 Bigelow PATENT201703001 Mar. 17, 2017 Bigelow PATENT201703002Mar. 24, 2017 Bigelow PATENT201703003 Mar. 24, 2017 BigelowPATENT201703004 Mar. 24, 2017 NRRL B-67264 May 16, 2016 NRRL B-67265 May16, 2016 NRRL B-67266 May 16, 2016 NRRL B-67267 May 16, 2016 NRRLB-67268 May 16, 2016 NRRL B-67269 May 16, 2016 NRRL B-67270 May 16, 2016

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as, an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

1. A method for decreasing incidence of mortality in a fowl, the methodcomprising: (a) administering to the fowl an effective amount of aprobiotic fowl supplement comprising: (i) a Clostridium sp., and (ii) aClostridium cluster XIV bacterium; and (b) a carrier suitable for fowladministration; wherein the fowl administered the effective amount ofthe probiotic fowl supplement exhibits a decrease in the incidence ofmortality, as compared to fowl not having been administered thesupplement.
 2. The method of claim 1, wherein the probiotic fowlsupplement further comprises a Bacillus sp.
 3. The method of claim 1,wherein the administering of the probiotic fowl supplement is an oraladministration.
 4. The method of claim 3, wherein the oraladministration comprises combining the probiotic fowl supplement withthe fowl's feed, water, litter, medicine, or vaccine; or spraying thefowl with the probiotic fowl supplement.
 5. The method of claim 1,wherein the Clostridium sp. and/or the Clostridium cluster XIV bacteriumare in spore form.
 6. The method of claim 1, wherein the probiotic fowlsupplement is administered to the fowl at least once a day.
 7. Themethod of claim 1, wherein each administration of the probiotic fowlsupplement comprises at least 10³ of the Clostridium sp.
 8. The methodof claim 1, wherein the fowl is a broiler.
 9. The method of claim 1,wherein (i) and/or (ii) are desiccated.
 10. The method of claim 9,wherein (i) and/or (ii) are freeze dried.
 11. The method of claim 9,wherein the probiotic fowl supplement has a moisture content of lessthan 10%.
 12. The method of claim 1, wherein the probiotic fowlsupplement is formulated as a water additive, a feed additive, apre-pelleted feed additive, a pelleted feed additive, apost-pelleting-applied feed additive, a consumable liquid, a consumablesolid, a consumable gel, or a gavage.
 13. The method of claim 12,wherein the probiotic fowl supplement occurs within or on pelleted feed.14. The method of claim 1, wherein the mortality is caused by a pathogenselected from: Mycoplasma gallisepticum, Mycoplasma meleagridis,Mycoplasma synoviae, Pasteurella multocida, Clostridium perfringens,Clostridium colinum, Clostridium botulinum, Salmonella typi, Salmonellatyphimurium, Salmonella enterica, Salmonella pullorum, Salmonellagallinarum, Haemophilus gallinarum, Erysipelothrix insidiosa,Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Listeriamonocytogenes, Arcobacter butzleri, Mycobacterium avium, and pathogenicstrains of Escherichia coli and Staphylococcus aureus.
 15. The method ofclaim 14, wherein the mortality is caused by Clostridium perfringens.16. The method of claim 1, wherein the incidence of mortality isdecreased by at least 5%.
 17. The method of claim 1, wherein theincidence of mortality is decreased by at least 10%.
 18. The method ofclaim 1, wherein the administration of the probiotic fowl supplementshifts the non-pathogenic gut microbiome of the fowl.
 19. The method ofclaim 18, wherein the gut microbiome shift is an increase in therelative abundance of one or more microbes that were present in the gutprior to administration of the probiotic fowl supplement.
 20. The methodof claim 18, wherein the gut microbiome shift is an increase in therelative abundance of obligate anaerobes and/or lactic acid-producingmicrobes.